Anti-IGF-I receptor antibody

ABSTRACT

Antibodies, humanized antibodies, resurfaced antibodies, antibody fragments, derivatized antibodies, and conjugates of same with cytotoxic agents, which specifically bind to, and inhibit, insulin-like growth factor-I receptor, antagonize the effects of IGF-I, IGF-II and serum on the growth and survival of tumor cells, and which are substantially devoid of agonist activity. Said antibodies and fragments thereof may be used, optionally in conjunction with other therapeutic agents, in the treatment of tumors that express elevated levels of IGF-I receptor, such as breast cancer, colon cancer, lung cancer, ovarian carcinoma, synovial sarcoma, prostate cancer and pancreatic cancer, and said derivatized antibodies may be used in the diagnosis and imaging of tumors that express elevated levels of IGF-I receptor.

The present application is a continuation-in-part of parent applicationSer. No. 10/170,390, filed Jun. 14, 2002, incorporated herein byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to antibodies that bind to humaninsulin-like growth factor-I receptor (IGF-I receptor). Moreparticularly, the invention relates to anti-IGF-I receptor antibodiesthat inhibit the cellular functions of the IGF-I receptor. Still moreparticularly, the invention relates to anti-IGF-I receptor antibodiesthat antagonize the effects of IGF-I, IGF-II and serum on the growth andsurvival of tumor cells and which are substantially devoid of agonistactivity. The invention also relates to fragments of said antibodies,humanized and resurfaced versions of said antibodies, conjugates of saidantibodies, antibody derivatives, and the uses of same in diagnostic,research and therapeutic applications. The invention further relates toimproved antibodies or fragments thereof that are made from theabove-described antibodies and fragments thereof. In another aspect, theinvention relates to a polynucleotide encoding the antibodies orfragments thereof, and to vectors comprising the polynucleotides.

BACKGROUND OF THE INVENTION

Insulin-like growth factor-I receptor (IGF-I receptor) is atransmembrane heterotetrameric protein, which has two extracellularalpha chains and two membrane-spanning beta chains in a disulfide-linkedβ-α-α-β configuration. The binding of the ligands, which areinsulin-like growth-factor-I (IGF-I) and insulin-like growth factor-II(IGF-II), by the extracellular domain of IGF-I receptor activates itsintracellular tyrosine kinase domain resulting in autophosphorylation ofthe receptor and substrate phosphorylation. The IGF-I receptor ishomologous to insulin receptor, having a high sequence similarity of 84%in the beta chain tyrosine kinase domain and a low sequence similarityof 48% in the alpha chain extracellular cysteine rich domain (Ulrich, A.et al., 1986, EMBO, 5, 2503-2512; Fujita-Yamaguchi, Y. et al., 1986, J.Biol. Chem., 261, 16727-16731; LeRoith, D. et al., 1995, EndocrineReviews, 16, 143-163). The IGF-I receptor and its ligands (IGF-I andIGF-II) play important roles in numerous physiological processesincluding growth and development during embryogenesis, metabolism,cellular proliferation and cell differentiation in adults (LeRoith, D.,2000, Endocrinology, 141, 1287-1288; LeRoith, D., 1997, New England J.Med., 336, 633-640).

IGF-I and IGF-II function both as endocrine hormones in the blood, wherethey are predominantly present in complexes with IGF-binding proteins,and as paracrine and autocrine growth factors that are produced locally(Humbel, R. E., 1990, Eur. J. Biochem., 190, 445-462; Cohick, W. S. andClemmons, D. R., 1993, Annu. Rev. Physiol. 55, 131-153).

The IGF-I receptor has been implicated in promoting growth,transformation and survival of tumor cells (Baserga, R. et al., 1997,Biochem. Biophys. Acta, 1332, F105-F126; Blakesley, V. A. et al., 1997,Journal of Endocrinology, 152, 339-344; Kaleko, M., Rutter, W. J., andMiller, A. D. 1990, Mol. Cell. Biol., 10, 464-473). Thus, several typesof tumors are known to express higher than normal levels of IGF-Ireceptor, including breast cancer, colon cancer, ovarian carcinoma,synovial sarcoma and pancreatic cancer (Khandwala, H. M. et al., 2000,Endocrine Reviews, 21, 215-244; Werner, H. and LeRoith, D., 1996, Adv.Cancer Res., 68,183-223; Happerfield, L. C. et al., 1997, J. Pathol.,183, 412-417; Frier, S. et al., 1999, Gut, 44, 704-708; van Dam, P. A.et al., 1994, J. Clin. Pathol., 47, 914-919; Xie, Y. et al., 1999,Cancer Res., 59, 3588-3591; Bergmann, U. et al., 1995, Cancer Res., 55,2007-2011). In vitro, IGF-I and IGF-II have been shown to be potentmitogens for several human tumor cell lines such as lung cancer, breastcancer, colon cancer, osteosarcoma and cervical cancer (Ankrapp, D. P.and Bevan, D. R., 1993, Cancer Res., 53, 3399-3404; Cullen, K. J., 1990,Cancer Res., 50, 48-53; Hermanto, U. et al., 2000, Cell Growth &Differentiation, 11, 655-664; Guo, Y. S. et al., 1995, J. Am. Coll.Surg., 181, 145-154; Kappel, C. C. et al., 1994, Cancer Res., 54,2803-2807; Steller, M. A. et al., 1996, Cancer Res., 56, 1761-1765).Several of these tumors and tumor cell lines also express high levels ofIGF-I or IGF-II, which may stimulate their growth in an autocrine orparacrine manner (Quinn, K. A. et al., 1996, J. Biol. Chem., 271,11477-11483).

Epidemiological studies have shown a correlation of elevated plasmalevel of IGF-I (and lower level of IGF-binding protein-3) with increasedrisk for prostate cancer, colon cancer, lung cancer and breast cancer(Chan, J. M. et al., 1998, Science, 279, 563-566; Wolk, A. et al., 1998,J. Natl. Cancer Inst., 90, 911-915; Ma, J. et al., 1999, J. Natl. CancerInst., 91, 620-625; Yu, H. et al., 1999, J. Natl. Cancer Inst., 91,151-156; Hankinson, S. E. et al., 1998, Lancet, 351, 1393-1396).Strategies to lower the IGF-I level in plasma or to inhibit the functionof IGF-I receptor have been suggested for cancer prevention (Wu, Y. etal., 2002, Cancer Res., 62, 1030-1035; Grimberg, A and Cohen P., 2000,J. Cell. Physiol., 183, 1-9).

The IGF-I receptor protects tumor cells from apoptosis caused by growthfactor deprivation, anchorage independence or cytotoxic drug treatment(Navarro, M. and Baserga, R., 2001, Endocrinology, 142, 1073-1081;Baserga, R. et al., 1997, Biochem. Biophys. Acta, 1332, F105-F126). Thedomains of IGF-I receptor that are critical for its mitogenic,transforming and anti-apoptotic activities have been identified bymutational analysis.

For example, the tyrosine 1251 residue of IGF-I receptor has beenidentified as critical for anti-apoptotic and transformation activitiesbut not for its mitogenic activity (O'Connor, R. et al., 1997, Mol.Cell. Biol., 17, 427-435; Miura, M. et al., 1995, J. Biol. Chem., 270,22639-22644). The intracellular signaling pathway of ligand-activatedIGF-I receptor involves phosphorylation of tyrosine residues of insulinreceptor substrates (IRS-1 and IRS-2), which recruitphosphatidylinositol-3-kinase (PI-3-kinase) to the membrane. Themembrane-bound phospholipid products of PI-3-kinase activate aserine/threonine kinase Akt, whose substrates include the pro-apoptoticprotein BAD which is phosphorylated to an inactive state (Datta, S. R.,Brunet, A. and Greenberg, M. E., 1999, Genes & Development, 13,2905-2927; Kulik, G., Klippel, A. and Weber, M. J., 1997, Mol. Cell.Biol. 17, 1595-1606). The mitogenic signaling of IGF-I receptor in MCF-7human breast cancer cells requires PI-3-kinase and is independent ofmitogen-activated protein kinase, whereas the survival signaling indifferentiated rat pheochromocytoma PC12 cells requires both PI-3-kinaseand mitogen-activated protein kinase pathways (Dufourny, B. et al.,1997, J. Biol. Chem., 272, 31163-31171; Parrizas, M., Saltiel, A. R. andLeRoith, D., 1997, J. Biol. Chem., 272, 154-161).

Down-regulation of IGF-I receptor level by anti-sense strategies hasbeen shown to reduce the tumorigenicity of several tumor cell lines invivo and in vitro, such as melanoma, lung carcinoma, ovarian cancer,glioblastoma, neuroblastoma and rhabdomyosarcoma (Resnicoff, M. et al.,1994, Cancer Res., 54, 4848-4850; Lee, C.-T. et al., 1996, Cancer Res.,56, 3038-3041; Muller, M. et al., 1998, Int. J. Cancer, 77, 567-571;Trojan, J. et al., 1993, Science, 259, 94-97; Liu, X. et al., 1998,Cancer Res., 58, 5432-5438; Shapiro, D. N. et al., 1994, J. Clin.Invest., 94, 1235-1242). Likewise, a dominant negative mutant of IGF-Ireceptor has been reported to reduce the tumorigenicity in vivo andgrowth in vitro of transformed Rat-1 cells overexpressing IGF-I receptor(Prager, D. et al., 1994, Proc. Natl. Acad. Sci. USA, 91, 2181-2185).

Tumor cells expressing an antisense to the IGF-I receptor mRNA undergomassive apoptosis when injected into animals in biodiffusion chambers.This observation makes the IGF-I receptor an attractive therapeutictarget, based upon the hypothesis that tumor cells are more susceptiblethan normal cells to apoptosis by inhibition of IGF-I receptor(Resnicoff, M. et al., 1995, Cancer Res., 55, 2463-2469; Baserga, R.,1995, Cancer Res., 55, 249-252).

Another strategy to inhibit the function of IGF-I receptor in tumorcells has been to use anti-IGF-I receptor antibodies which bind to theextracellular domains of IGF-I receptor and inhibit its activation.Several attempts have been reported to develop mouse monoclonalantibodies against IGF-I receptor, of which two inhibitoryantibodies—IR3 and 1H7—are available and their use has been reported inseveral IGF-I receptor studies.

The IR3 antibody was developed using a partially purified placentalpreparation of insulin receptor to immunize mice, which yielded anantibody, IR1, that was selective for binding insulin receptor, and twoantibodies, IR2 and IR3, that showed preferential immunoprecipitation ofIGF-I receptor (somatomedin-C receptor) but also weakimmunoprecipitation of insulin receptor (Kull, F. C. et al., 1983, J.Biol. Chem., 258, 6561-6566).

The 1H7 antibody was developed by immunizing mice with purifiedplacental preparation of IGF-I receptor, which yielded the inhibitoryantibody 1H7 in addition to three stimulatory antibodies (Li, S.-L. etal., 1993, Biochem. Biophys. Res. Commun., 196, 92-98; Xiong, L. et al.,1992, Proc. Natl. Acad. Sci. USA, 89, 5356-5360).

In another report, a panel of mouse monoclonal antibodies specific forhuman IGF-I receptor were obtained by immunization of mice withtransfected 3T3 cells expressing high levels of IGF-I receptor, whichwere categorized into seven groups by binding competition studies and bytheir inhibition or stimulation of IGF-I binding to transfected 3T3cells (Soos, M. A. et al., 1992, J. Biol. Chem., 267, 12955-12963).

Thus, although IR3 antibody is the most commonly used inhibitoryantibody for IGF-I receptor studies in vitro, it suffers from thedrawback that it exhibits agonistic activity in transfected 3T3 and CHOcells expressing human IGF-I receptor (Kato, H. et al., 1993, J. Biol.Chem., 268, 2655-2661; Steele-Perkins, G. and Roth, R. A., 1990,Biochem. Biophys. Res. Commun., 171, 1244-1251). Similarly, among thepanel of antibodies developed by Soos et al., the most inhibitoryantibodies 24-57 and 24-60 also showed agonistic activities in thetransfected 3T3 cells (Soos, M. A. et al., 1992, J. Biol. Chem., 267,12955-12963). Although, IR3 antibody is reported to inhibit the bindingof IGF-I (but not IGF-II) to expressed receptors in intact cells andafter solubilization, it is shown to inhibit the ability of both IGF-Iand IGF-II to stimulate DNA synthesis in cells in vitro (Steele-Perkins,G. and Roth, R. A., 1990, Biochem. Biophys. Res. Commun., 171,1244-1251). The binding epitope of IR3 antibody has been inferred fromchimeric insulin-IGF-I receptor constructs to be the 223-274 region ofIGF-I receptor (Gustafson, T. A. and Rutter, W. J., 1990, J. Biol.Chem., 265, 18663-18667; Soos, M. A. et al., 1992, J. Biol. Chem., 267,12955-12963).

The MCF-7 human breast cancer cell line is typically used as a modelcell line to demonstrate the growth response of IGF-I and IGF-II invitro (Dufourny, B. et al., 1997, J. Biol. Chem., 272, 31163-31171). InMCF-7 cells, the IR3 antibody incompletely blocks the stimulatory effectof exogenously added IGF-I and IGF-II in serum-free conditions byapproximately 80%. Also, the IR3 antibody does not significantly inhibit(less than 25%) the growth of MCF-7 cells in 10% serum (Cullen, K. J. etal., 1990, Cancer Res., 50, 48-53). This weak inhibition ofserum-stimulated growth of MCF-7 cells by IR3 antibody in vitro may berelated to the results of an in vivo study in which IR3 antibodytreatment did not significantly inhibit the growth of a MCF-7 xenograftin nude mice (Arteaga, C. L. et al., 1989, J. Clin. Invest., 84,1418-1423).

Because of the weak agonistic activities of the IR3 and other reportedantibodies, and their inability to significantly inhibit the growth oftumor cells such as MCF-7 cells in the more physiological condition ofserum-stimulation (instead of stimulation by exogenously added IGF-I orIGF-II in serum-free condition), there is a need for new anti-IGF-Ireceptor antibodies which significantly inhibit the serum-stimulatedgrowth of tumor cells but which do not show significant agonisticactivity by themselves.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the invention to provide antibodies,antibody fragments and antibody derivatives that specifically bind toinsulin-like growth factor-I receptor and inhibit the cellular activityof the receptor by antagonizing the receptor, and are also substantiallydevoid of agonist activity towards the receptor.

Thus, in a first embodiment, there is provided murine antibody EM164,which is fully characterized herein with respect to the amino acidsequences of both its light and heavy chain variable regions, the cDNAsequences of the genes for the light and heavy chain variable regions,the identification of its CDRs (complementarity-determining regions),the identification of its surface amino acids, and means for itsexpression in recombinant form.

In a second embodiment, there are provided resurfaced or humanizedversions of antibody EM164 wherein surface-exposed residues of theantibody or its fragments are replaced in both light and heavy chains tomore closely resemble known human antibody surfaces. Such humanizedantibodies may have increased utility, compared to murine EM164, astherapeutic or diagnostic agents. Humanized versions of antibody EM164are also fully characterized herein with respect to their respectiveamino acid sequences of both light and heavy chain variable regions, theDNA sequences of the genes for the light and heavy chain variableregions, the identification of the CDRs, the identification of theirsurface amino acids, and disclosure of a means for their expression inrecombinant form.

In a third embodiment, there is provided an antibody that is capable ofinhibiting the growth of a cancer cell by greater than about 80% in thepresence of a growth stimulant such as, for example, serum, insulin-likegrowth factor-I and insulin-like growth factor-II.

In a fourth embodiment, there is provided an antibody or antibodyfragment having a heavy chain including CDRs having amino acid sequencesrepresented by SEQ ID NOS: 1-3, respectively: SYWMH: (SEQ ID NO:1)EINPSNGRTNYNEKFKR, (SEQ ID NO:2) GRPDYYGSSKWYFDV; (SEQ ID NO:3)

and having a light chain that comprises CDRs having amino acid sequencesrepresented by SEQ ID NOS:4-6: RSSQSIVHSNVNTYLE; (SEQ ID NO:4) KVSNRFS;(SEQ ID NO:5) FQGSHVPPT. (SEQ ID NO:6)

In a fifth embodiment, there are provided antibodies having a heavychain that has an amino acid sequence that shares at least 90% sequenceidentity with an amino acid sequence represented by SEQ ID NO:7: (SEQ IDNO:7) QVQLQQSGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPSNGRTNYNEKFKRKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSSKWYFDVWGAGTTVTVSS.

Similarly, there are provided antibodies having a light chain that hasan amino acid sequence that shares at least 90% sequence identity withan amino acid sequence represented by SEQ ID NO:8: (SEQ ID NO:8)DVLMTQTPLSLPVSLGDQASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAEDLGIYYCFQGSHVP PTFGGGTKLEIKR.

In a sixth embodiment, antibodies are provided having a humanized orresurfaced light chain variable region having an amino acid sequencecorresponding to one of SEQ ID NOS:9-12: (SEQ ID NO:9)DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP PTFGGGTKLEIKR; (SEQID NO:10) DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP PTFGGGTKLEIKR; (SEQID NO:11) DVLMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP PTFGGGTKLEIKR; or(SEQ ID NO:12) DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVP PTFGGGTKLEIKR.

Similarly, antibodies are provided having a humanized or resurfacedheavy chain variable region having an amino acid sequence correspondingto SEQ ID NO: 13: (SEQ ID NO:13)QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPDYYGSSKWYFDVWGQGTTVTVSS.

In a seventh embodiment, antibodies or antibody fragments of the presentinvention are provided that have improved properties. For example,antibodies or antibody fragments having improved affinity forIGF-I-receptor are prepared by affinity maturation of an antibody orfragment of the present invention.

The present invention further provides conjugates of said antibodies,wherein a cytotoxic agent is covalently attached, directly or via acleavable or non-cleavable linker, to an antibody or epitope-bindingfragment of an antibody of the present invention. In preferredembodiments, the cytotoxic agent is a taxol, a maytansinoid, CC-1065 ora CC-1065 analog.

The present invention further provides for antibodies or fragmentsthereof that are further labeled for use in research or diagnosticapplications. In preferred embodiments, the label is a radiolabel, afluorophore, a chromophore, an imaging agent or a metal ion.

A method for diagnosis is also provided in which said labeled antibodiesor fragments are administered to a subject suspected of having a cancer,and the distribution of the label within the body of the subject ismeasured or monitored.

In an eighth embodiment, the invention provides methods for thetreatment of a subject having a cancer by administering an antibody,antibody fragment or antibody conjugate of the present invention, eitheralone or in combination with other cytotoxic or therapeutic agents. Thecancer can be one or more of, for example, breast cancer, colon cancer,ovarian carcinoma, osteosarcoma, cervical cancer, prostate cancer, lungcancer, synovial carcinoma, pancreatic cancer, or other cancer yet to bedetermined in which IGF-I receptor levels are elevated.

In a ninth embodiment, the invention provides methods for the treatmentof a subject having a cancer by administering an antibody, antibodyfragment or antibody conjugate of the present invention, either alone orin combination with other cytotoxic or therapeutic agents. Inparticular, preferred cytotoxic and therapeutic agents includedocetaxel, paclitaxel, doxorubicin, epirubicin, cyclophosphamide,trastuzumab (Herceptin), capecitabine, tamoxifen, toremifene, letrozole,anastrozole, fulvestrant, exemestane, goserelin, oxaliplatin,carboplatin, cisplatin, dexamethasone, antide, bevacizumab (Avastin),5-fluorouracil, leucovorin, levamisole, irinotecan, etoposide,topotecan, gemcitabine, vinorelbine, estramustine, mitoxantrone,abarelix, zoledronate, streptozocin, rituximab (Rituxan), idarubicin,busulfan, chlorambucil, fludarabine, imatinib, cytarabine, ibritumomab(Zevalin), tositumomab (Bexxar), interferon alpha-2b, melphalam,bortezomib (Velcade), altretamine, asparaginase, gefitinib (Iressa),erlonitib (Tarceva), anti-EGF receptor antibody (Cetuximab, Abx-EGF),and an epothilone. More preferably, the therapeutic agent is a platinumagent (such as carboplatin, oxaliplatin, cisplatin), a taxane (such aspaclitaxel, docetaxel), gemcitabine, or camptothecin.

The cancer can be one or more of, for example, breast cancer, coloncancer, ovarian carcinoma, osteosarcoma, cervical cancer, prostatecancer, lung cancer, synovial carcinoma, pancreatic cancer, melanoma,multiple myeloma, neuroblastoma, and rhabdomyosarcoma, or other canceryet to be determined in which IGF-I receptor levels are elevated.

In a tenth embodiment, the invention provides kits comprising one ormore of the elements described herein, and instructions for the use ofthose elements. In a preferred embodiment, a kit of the presentinvention includes antibody, antibody fragment or conjugate of theinvention, and a therapeutic agent. The instructions for this preferredembodiment include instructions for inhibiting the growth of a cancercell using the antibody, antibody fragment or conjugate of theinvention, and the therapeutic agent, and/or instructions for a methodof treating a patient having a cancer using the antibody, antibodyfragment or conjugate of the invention, and the therapeutic agent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows fluorescence activated cell sorting (FACS) analysis of thespecific binding of purified EM164 antibody to cells overexpressinghuman Y1251F IGF-I receptor or human insulin receptor.

FIG. 2 shows a binding titration curve for the binding of EM164 antibodyto biotinylated human IGF-I receptor.

FIG. 3 shows the inhibition of the binding of biotinylated IGF-I tohuman breast cancer MCF-7 cells by EM164 antibody.

FIG. 4 shows the inhibition of IGF-I-stimulated autophosphorylation ofIGF-I receptor in MCF-7 cells by EM164 antibody.

FIG. 5 shows the inhibition of IGF-I-stimulated IRS-1-phosphorylation inMCF-7 cells by EM164 antibody.

FIG. 6 shows the inhibition of IGF-I-stimulated signal transduction inSaOS-2 cells by EM164 antibody.

FIG. 7 shows the effect of EM164 antibody on the growth and survival ofMCF-7 cells under different growth conditions, as assessed by MTT assay.

FIG. 8 shows the effect of EM164 antibody on the growth and survival ofMCF-7 cells in the presence of various serum concentrations.

FIG. 9 shows the inhibition of IGF-I- and serum-stimulated growth andsurvival of NCI-H838 cells by EM164 antibody.

FIG. 10 shows the effect of treatment with EM164 antibody, taxol, or acombination of EM164 antibody and taxol, on the growth of a Calu-6 lungcancer xenograft in mice.

FIG. 11 shows competition between the binding of humanized EM164antibody (v.1.0) and murine EM164 antibody.

FIG. 12 shows the cDNA (SEQ ID NO:49) and amino acid sequences (SEQ IDNO:50) of the light chain leader and variable region of the murineanti-IGF-I receptor antibody EM164. The arrow marks the start offramework 1. The 3 CDR sequences according to Kabat are underlined.

FIG. 13 shows the cDNA (SEQ ID NO:51) and amino acid sequences (SEQ IDNO:52) of the heavy chain leader and variable region for the murineanti-IGF-I receptor antibody EM164. The arrow marks the start offramework 1. The 3 CDR sequences according to Kabat are underlined.

FIG. 14 shows the light and heavy chain CDR amino acid sequences ofantibody EM164 as determined from Chothia canonical class definitions.AbM modeling software definitions for the heavy chain CDRs are alsoshown. Light Chain: CDR1 is SEQ ID NO:4, CDR2 is SEQ ID NO:5, and CDR3is SEQ ID NO:6. Heavy Chain: CDR1 is SEQ ID NO:1, CDR2 is SEQ ID NO:2,and CDR3 is SEQ ID NO:3. AbM Heavy Chain: CDR1 is SEQ ID NO:53, CDR2 isSEQ ID NO:54, and CDR3 is SEQ ID NO:55.

FIG. 15 shows the light chain and heavy chain amino acid sequences foranti-IGF-I-receptor antibody EM164 aligned with the germline sequencesfor the Cr1 (SEQ ID NO:56) and J558.c (SEQ ID NO:57) genes. Dashes (-)indicate sequence identity.

FIG. 16 shows the plasmids used to build and express the recombinantchimeric and humanized EM164 antibodies. A) a light chain cloningplasmid, B) a heavy chain cloning plasmid, C) a mammalian antibodyexpression plasmid.

FIG. 17 shows the 10 most homologous amino acid sequences of the lightchains screened from the 127 antibodies in the set of structure filesused to predict the surface residues of EM164. em164 LC (SEQ ID NO:58),2jel (SEQ ID NO:59), 2pcp (SEQ ID NO:60), 1nqb (SEQ ID NO:61), 1kel (SEQID NO:62), 1hyx (SEQ ID NO:63), 1igf (SEQ ID NO:64), 1tet (SEQ IDNO:65), 1clz (SEQ ID NO:66), 1bln (SEQ ID NO:67), 1cly (SEQ ID NO:68),Consensus (SEQ ID NO:69).

FIG. 18 shows the 10 most homologous amino acid sequences of the heavychains screened from the 127 antibodies in the set of structure filesused to predict the surface residues of EM164. em164 HC (SEQ ID NO:70),1nqb (SEQ ID NO:71), 1ngp (SEQ ID NO:72), 1fbi (SEQ ID NO:73), 1afv (SEQID NO:74), 1yuh (SEQ ID NO:75), 1plg (SEQ ID NO:76), 1d5b (SEQ IDNO:77), 1ae6 (SEQ ID NO:78), 1axs (SEQ ID NO:79), 3hfl (SEQ ID NO:80),Consensus (SEQ ID NO:81).

FIG. 19 shows the average accessibility for each of the (A) light, and(B) heavy chain variable region residues from the 10 most homologousstructures. The numbers represent the Kabat antibody sequence positionnumbers.

FIG. 20 shows the light chain variable region amino acid sequences formurine EM164 (muEM164) and humanized EM164 (huEM164) antibodies. muEM164(SEQ ID NO:82), huEM164 V1.0 (SEQ ID NO:83), huEM164 V1.1 (SEQ IDNO:84), huEM164 V1.2 (SEQ ID NO:85), huEM164 V1.3 (SEQ ID NO:86).

FIG. 21 shows the heavy chain variable region amino acid sequences formurine (muEM164, SEQ ID NO:87) and humanized EM164 antibodies (huEM164,SEQ ID NO:88).

FIG. 22 shows the huEM164 v1.0 variable region DNA and amino acidsequences for both the light (DNA, SEQ ID NO:89, amino acid SEQ IDNO:90) and heavy chains (DNA, SEQ ID NO:91, amino acid SEQ ID NO:92).

FIG. 23 shows the light chain variable region DNA and amino acidsequences for humanized EM164 v1.1 (DNA, SEQ ID NO:93; amino acid SEQ IDNO:94), v1.2 (DNA, SEQ ID NO:95; amino acid SEQ ID NO:96) and v1.3 (DNA,SEQ ID NO:97; amino acid SEQ ID NO:98).

FIG. 24 shows the inhibition of IGF-I-stimulated growth and survival ofMCF-7 cells by humanized EM164 v1.0 antibody and murine EM164 antibody.

FIG. 25 shows that EM164 suppresses IGF-1-stimulated cycling of MCF-7cells.

FIG. 26 shows that EM164 suppresses the anti-apoptotic effect of IGF-1and serum. Treatment with EM164 results in apoptotic cell death asdemonstrated by the increased levels of cleaved CK18 protein.

FIG. 27 shows the effect of treatment with EM164 antibody, gemcitabine,or a combination of EM164 antibody and gemcitabine, on the growth ofhuman BxPC-3 pancreatic cancer xenografts in immunodeficient mice.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have discovered and improved novel antibodies thatspecifically bind to the human insulin-like growth factor-I receptor(IGF-IR) on the cell surface. The antibodies and fragments have theunique ability to inhibit the cellular functions of the receptor withoutthe capacity to activate the receptor themselves. Thus, while previouslyknown antibodies that specifically bind and inhibit IGF-IR also activatethe receptor even in the absence of IGF-IR ligands, the antibodies orfragments of the present invention antagonize IGF-IR but aresubstantially devoid of agonist activity. Furthermore, the antibodiesand antibody fragments of the present invention inhibit the growth ofhuman tumor cells such as MCF-7 cells in the presence of serum bygreater than 80%, which is a higher degree of inhibition than isobtained using previously known anti-IGF-IR antibodies.

The present invention proceeds from a murine anti-IGF-IR antibody,herein EM164, that is fully characterized with respect to the amino acidsequences of both light and heavy chains, the identification of theCDRs, the identification of surface amino acids, and means for itsexpression in recombinant form.

The germline sequences are shown in FIG. 15 aligned with the sequence ofEM164. The comparison identifies probable somatic mutations in EM164,including one each in CDR1 in the light chain and in CDR2 in the heavychain.

The primary amino acid and DNA sequences of antibody EM164 light andheavy chains, and of humanized versions, are disclosed herein. However,the scope of the present invention is not limited to antibodies andfragments comprising these sequences. Instead, all antibodies andfragments that specifically bind to an insulin-like growth factor-Ireceptor and antagonize the biological activity of the receptor, butwhich are substantially devoid of agonist activity, fall within thescope of the present invention. Thus, antibodies and antibody fragmentsmay differ from antibody EM164 or the humanized derivatives in the aminoacid sequences of their scaffold, CDRs, light chain and heavy chain, andstill fall within the scope of the present invention.

The CDRs of antibody EM164 are identified by modeling and theirmolecular structures have been predicted. Again, while the CDRs areimportant for epitope recognition, they are not essential to theantibodies and fragments of the invention. Accordingly, antibodies andfragments are provided that have improved properties produced by, forexample, affinity maturation of an antibody of the present invention.

Diverse antibodies and antibody fragments, as well as antibody mimicsmay be readily produced by mutation, deletion and/or insertion withinthe variable and constant region sequences that flank a particular setof CDRs. Thus, for example, different classes of Ab are possible for agiven set of CDRs by substitution of different heavy chains, whereby,for example, IgG1-4, IgM, IgA1-2, IgD, IgE antibody types and isotypesmay be produced. Similarly, artificial antibodies within the scope ofthe invention may be produced by embedding a given set of CDRs within anentirely synthetic framework. The term “variable” is used herein todescribe certain portions of the variable domains that differ insequence among antibodies and are used in the binding and specificity ofeach particular antibody for its antigen. However, the variability isnot usually evenly distributed through the variable domains of theantibodies. It is typically concentrated in three segments calledcomplementarity determining regions (CDRs) or hypervariable regions bothin the light chain and the heavy chain variable domains. The more highlyconserved portions of the variable domains are called the framework(FR). The variable domains of heavy and light chains each comprise fourframework regions, largely adopting a beta-sheet configuration,connected by three CDRs, which form loops connecting, and in some casesforming part of the beta-sheet structure. The CDRs in each chain areheld together in close proximity by the FR regions and, with the CDRsfrom the other chain, contribute to the formation of the antigen bindingsite of antibodies (E. A. Kabat et al. Sequences of Proteins ofImmunological Interest, fifth edition, 1991, NIH). The constant domainsare not involved directly in binding an antibody to an antigen, butexhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

Humanized antibodies, or antibodies adapted for non-rejection by othermammals, may be produced using several technologies such as resurfacingand CDR grafting. In the resurfacing technology, molecular modeling,statistical analysis and mutagenesis are combined to adjust the non-CDRsurfaces of variable regions to resemble the surfaces of knownantibodies of the target host. Strategies and methods for theresurfacing of antibodies, and other methods for reducing immunogenicityof antibodies within a different host, are disclosed in U.S. Pat. No.5,639,641, which is hereby incorporated in its entirety by reference. Inthe CDR grafting technology, the murine heavy and light chain CDRs aregrafted into a fully human framework sequence.

The invention also includes functional equivalents of the antibodiesdescribed in this specification. Functional equivalents have bindingcharacteristics that are comparable to those of the antibodies, andinclude, for example, chimerized, humanized and single chain antibodiesas well as fragments thereof. Methods of producing such functionalequivalents are disclosed in PCT Application WO 93/21319, EuropeanPatent Application No. 239,400; PCT Application WO 89/09622; EuropeanPatent Application 338,745; and European Patent Application EP 332,424,which are incorporated in their respective entireties by reference.

Functional equivalents include polypeptides with amino acid sequencessubstantially the same as the amino acid sequence of the variable orhypervariable regions of the antibodies of the invention. “Substantiallythe same” as applied to an amino acid sequence is defined herein as asequence with at least about 90%, and more preferably at least about 95%sequence identity to another amino acid sequence, as determined by theFASTA search method in accordance with Pearson and Lipman, Proc. Natl.Acad. Sci. USA 85, 2444-2448 (1988).

Chimerized antibodies preferably have constant regions derivedsubstantially or exclusively from human antibody constant regions andvariable regions derived substantially or exclusively from the sequenceof the variable region from a mammal other than a human. Humanized formsof the antibodies are made by substituting the complementaritydetermining regions of, for example, a mouse antibody, into a humanframework domain, e.g., see PCT Pub. No. WO92/22653. Humanizedantibodies preferably have constant regions and variable regions otherthan the complementarity determining regions (CDRs) derivedsubstantially or exclusively from the corresponding human antibodyregions and CDRs derived substantially or exclusively from a mammalother than a human.

Functional equivalents also include single-chain antibody fragments,also known as single-chain antibodies (scFvs). These fragments containat least one fragment of an antibody variable heavy-chain amino acidsequence (V_(H)) tethered to at least one fragment of an antibodyvariable light-chain sequence (V_(L)) with or without one or moreinterconnecting linkers. Such a linker may be a short, flexible peptideselected to assure that the proper three-dimensional folding of the(V_(L)) and (V_(H)) domains occurs once they are linked so as tomaintain the target molecule binding-specificity of the whole antibodyfrom which the single-chain antibody fragment is derived. Generally, thecarboxyl terminus of the (V_(L)) or (V_(H)) sequence may be covalentlylinked by such a peptide linker to the amino acid terminus of acomplementary (V_(L)) and (V_(H)) sequence. Single-chain antibodyfragments may be generated by molecular cloning, antibody phage displaylibrary or similar techniques. These proteins may be produced either ineukaryotic cells or prokaryotic cells, including bacteria.

Single-chain antibody fragments contain amino acid sequences having atleast one of the variable or complementarity determining regions (CDRs)of the whole antibodies described in this specification, but are lackingsome or all of the constant domains of those antibodies. These constantdomains are not necessary for antigen binding, but constitute a majorportion of the structure of whole antibodies. Single-chain antibodyfragments may therefore overcome some of the problems associated withthe use of antibodies containing a part or all of a constant domain. Forexample, single-chain antibody fragments tend to be free of undesiredinteractions between biological molecules and the heavy-chain constantregion, or other unwanted biological activity. Additionally,single-chain antibody fragments are considerably smaller than wholeantibodies and may therefore have greater capillary permeability thanwhole antibodies, allowing single-chain antibody fragments to localizeand bind to target antigen-binding sites more efficiently. Also,antibody fragments can be produced on a relatively large scale inprokaryotic cells, thus facilitating their production. Furthermore, therelatively small size of single-chain antibody fragments makes them lesslikely to provoke an immune response in a recipient than wholeantibodies.

Functional equivalents further include fragments of antibodies that havethe same, or comparable binding characteristics to those of the wholeantibody. Such fragments may contain one or both Fab fragments or theF(ab′)₂ fragment. Preferably the antibody fragments contain all sixcomplementarity determining regions of the whole antibody, althoughfragments containing fewer than all of such regions, such as three, fouror five CDRs, are also functional. Further, the functional equivalentsmay be or may combine members of any one of the following immunoglobulinclasses: IgG, IgM, IgA, IgD, or IgE, and the subclasses thereof.

The knowledge of the amino acid and nucleic acid sequences for theanti-IGF-I receptor antibody EM164 and its humanized variants, which aredescribed herein, can be used to develop other antibodies which alsobind to human IGF-I receptor and inhibit the cellular functions of theIGF-I receptor. Several studies have surveyed the effects of introducingone or more amino acid changes at various positions in the sequence ofan antibody, based on the knowledge of the primary antibody sequence, onits properties such as binding and level of expression (Yang, W. P. etal., 1995, J. Mol. Biol., 254, 392-403; Rader, C. et al., 1998, Proc.Natl. Acad. Sci. USA, 95, 8910-8915; Vaughan, T. J. et al., 1998, NatureBiotechnology, 16, 535-539).

In these studies, variants of the primary antibody have been generatedby changing the sequences of the heavy and light chain genes in theCDR1, CDR2, CDR3, or framework regions, using methods such asoligonucleotide-mediated site-directed mutagenesis, cassettemutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E.coli (Vaughan, T. J. et al., 1998, Nature Biotechnology, 16, 535-539;Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, in “Phage Display ofPeptides and Proteins”, Eds. Kay, B. K. et al., Academic Press). Thesemethods of changing the sequence of the primary antibody have resultedin improved affinities of the secondary antibodies (Gram, H. et al.,1992, Proc. Natl. Acad. Sci. USA, 89, 3576-3580; Boder, E. T. et al.,2000, Proc. Natl. Acad. Sci. USA, 97, 10701-10705; Davies, J. andRiechmann, L., 1996, Immunotechnolgy, 2, 169-179; Thompson, J. et al.,1996, J. Mol. Biol., 256, 77-88; Short, M. K. et al., 2002, J. Biol.Chem., 277, 16365-16370; Furukawa, K. et al., 2001, J. Biol. Chem., 276,27622-27628).

By a similar directed strategy of changing one or more amino acidresidues of the antibody, the antibody sequences described in thisinvention can be used to develop anti-IGF-I receptor antibodies withimproved functions.

The conjugates of the present invention comprise the antibody,fragments, and their analogs as disclosed herein, linked to a cytotoxicagent. Preferred cytotoxic agents are maytansinoids, taxanes and analogsof CC-1065. The conjugates can be prepared by in vitro methods. In orderto link the cytotoxic agent to the antibody, a linking group is used.Suitable linking groups are well known in the art and include disulfidegroups, thioether groups, acid labile groups, photolabile groups,peptidase labile groups and esterase labile groups. Preferred linkinggroups are disulfide groups and thioether groups. For example,conjugates can be constructed using a disulfide exchange reaction or byforming a thioether bond between the antibody and the cytotoxic agent.

Maytansinoids and maytansinoid analogs are among the preferred cytotoxicagents. Examples of suitable maytansinoids include maytansinol andmaytansinol analogs. Suitable maytansinoids are disclosed in U.S. Pat.Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929;4,331,598; 4,361,650; 4,362,663; 4,364,866; 4,450,254; 4,322,348;4,371,533; 6,333,410; 5,475,092; 5,585,499; and 5,846,545.

Taxanes are also preferred cytotoxic agents. Taxanes suitable for use inthe present invention are disclosed in U.S. Pat. Nos. 6,372,738 and6,340,701.

CC-1065 and its analogs are also preferred cytotoxic drugs for use inthe present invention. CC-1065 and its analogs are disclosed in U.S.Pat. Nos. 6,372,738; 6,340,701; 5,846,545 and 5,585,499.

An attractive candidate for the preparation of such cytotoxic conjugatesis CC-1065, which is a potent anti-tumor antibiotic isolated from theculture broth of Streptomyces zelensis. CC-1065 is about 1000-fold morepotent in vitro than are commonly used anti-cancer drugs, such asdoxorubicin, methotrexate and vincristine (B. K. Bhuyan et al., CancerRes., 42, 3532-3537 (1982)).

Cytotoxic drugs such as methotrexate, daunorubicin, doxorubicin,vincristine, vinblastine, melphalan, mitomycin C, chlorambucil, andcalicheamicin are also suitable for the preparation of conjugates of thepresent invention, and the drug molecules can also be linked to theantibody molecules through an intermediary carrier molecule such asserum albumin.

For diagnostic applications, the antibodies of the present inventiontypically will be labeled with a detectable moiety. The detectablemoiety can be any one which is capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹³¹I; a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase.

Any method known in the art for conjugating the antibody to thedetectable moiety may be employed, including those methods described byHunter, et al., Nature 144:945 (1962); David, et al., Biochemistry13:1014 (1974); Pain, et al., J. Immunol. Meth. 40:219 (1981); andNygren, J. Histochem. and Cytochem. 30:407 (1982).

The antibodies of the present invention can be employed in any knownassay method, such as competitive binding assays, direct and indirectsandwich assays, and immunoprecipitation assays (Zola, MonoclonalAntibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987)).

The antibodies of the invention also are useful for in vivo imaging,wherein an antibody labeled with a detectable moiety such as aradio-opaque agent or radioisotope is administered to a subject,preferably into the bloodstream, and the presence and location of thelabeled antibody in the host is assayed. This imaging technique isuseful in the staging and treatment of malignancies. The antibody may belabeled with any moiety that is detectable in a host, whether by nuclearmagnetic resonance, radiology, or other detection means known in theart.

The antibodies of the invention also are useful as affinity purificationagents. In this process, the antibodies are immobilized on a suitablesupport, such a Sephadex resin or filter paper, using methods well knownin the art.

The antibodies of the invention also are useful as reagents inbiological research, based on their inhibition of the function of IGF-Ireceptor in cells.

For therapeutic applications, the antibodies or conjugates of theinvention are administered to a subject, in a pharmaceuticallyacceptable dosage form. They can be administered intravenously as abolus or by continuous infusion over a period of time, by intramuscular,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. The antibody may also be administered byintratumoral, peritumoral, intralesional, or perilesional routes, toexert local as well as systemic therapeutic effects. Suitablepharmaceutically acceptable carriers, diluents, and excipients are wellknown and can be determined by those of skill in the art as the clinicalsituation warrants. Examples of suitable carriers, diluents and/orexcipients include: (1) Dulbecco's phosphate buffered saline, pH about7.4, containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. The method of thepresent invention can be practiced in vitro, in vivo, or ex vivo.

In other therapeutic treatments, the antibodies, antibody fragments orconjugates of the invention are co-administered, or administeredsequentially, with one or more additional therapeutic agents. Suitabletherapeutic agents include, but are not limited to, cytotoxic orcytostatic agents. Taxol is a preferred therapeutic agent that is also acytotoxic agent.

Cancer therapeutic agents are those agents that seek to kill or limitthe growth of cancer cells while doing minimal damage to the host. Thus,such agents may exploit any difference in cancer cell properties (e.g.metabolism, vascularization or cell-surface antigen presentation) fromhealthy host cells. Differences in tumor morphology are potential sitesfor intervention: for example, the second therapeutic can be an antibodysuch as an anti-VEGF antibody that is useful in retarding thevascularization of the interior of a solid tumor, thereby slowing itsgrowth rate. Other therapeutic agents include, but are not limited to,adjuncts such as granisetron HCl, androgen inhibitors such as leuprolideacetate, antibiotics such as doxorubicin, antiestrogens such astamoxifen, antimetabolites such as interferon alpha-2a, cytotoxic agentssuch as taxol, enzyme inhibitors such as ras famesyl-transferaseinhibitor, immunomodulators such as aldesleukin, and nitrogen mustardderivatives such as melphalan HCl, and the like.

The therapeutic agents that can be combined with EM164 for improvedanti-cancer efficacy include diverse agents used in oncology practice(Reference: Cancer, Principles & Practice of Oncology, DeVita, V. T.,Hellman, S., Rosenberg, S. A., 6th edition, Lippincott-Raven,Philadelphia, 2001), such as docetaxel, paclitaxel, doxorubicin,epirubicin, cyclophosphamide, trastuzumab (Herceptin), capecitabine,tamoxifen, toremifene, letrozole, anastrozole, fulvestrant, exemestane,goserelin, oxaliplatin, carboplatin, cisplatin, dexamethasone, antide,bevacizumab (Avastin), 5-fluorouracil, leucovorin, levamisole,irinotecan, etoposide, topotecan, gemcitabine, vinorelbine,estramustine, mitoxantrone, abarelix, zoledronate, streptozocin,rituximab (Rituxan), idarubicin, busulfan, chlorambucil, fludarabine,imatinib, cytarabine, ibritumomab (Zevalin), tositumomab (Bexxar),interferon alpha-2b, melphalam, bortezomib (Velcade), altretamine,asparaginase, gefitinib (Iressa), erlonitib (Tarceva), anti-EGF receptorantibody (Cetuximab, Abx-EGF), epothilones, and conjugates of cytotoxicdrugs and antibodies against cell-surface receptors. Preferredtherapeutic agents are platinum agents (such as carboplatin,oxaliplatin, cisplatin), taxanes (such as paclitaxel, docetaxel),gemcitabine, and camptothecin.

The one or more additional therapeutic agents can be administeredbefore, concurrently, or after the antibody, antibody fragment orconjugate of the invention. The skilled artisan will understand that foreach therapeutic agent there may be advantages to a particular order ofadministration. Similarly, the skilled artisan will understand that foreach therapeutic agent, the length of time between which the agent, andan antibody, antibody fragment or conjugate of the invention isadministered, will vary.

While the skilled artisan will understand that the dosage of eachtherapeutic agent will be dependent on the identity of the agent, thepreferred dosages can range from about 10 mg/square meter to about 2000mg/square meter, more preferably from about 50 mg/square meter to about1000 mg/square meter. For preferred agents such as platinum agents(carboplatin, oxaliplatin, cisplatin), the preferred dosage is about 10mg/square meter to about 400 mg/square meter, for taxanes (paclitaxel,docetaxel) the preferred dosage is about 20 mg/square meter to about 150mg/square meter, for gemcitabine the preferred dosage is about 100mg/square meter to about 2000 mg/square meter, and for camptothecin thepreferred dosage is about 50 mg/square meter to about 350 mg/squaremeter. The dosage of this and other therapeutic agents may depend onwhether the antibody, antibody fragment or conjugate of the invention isadministered concurrently or sequentially with a therapeutic agent.

Administration of an antibody, antibody fragment or conjugate of theinvention, and one or more additional therapeutic agents, whetherco-administered or administered sequentially, may occur as describedabove for therapeutic applications. Suitable pharmaceutically acceptablecarriers, diluents, and excipients for co-administration will beunderstood by the skilled artisan to depend on the identity of theparticular therapeutic agent being co-administered.

When present in an aqueous dosage form, rather than being lyophilized,the antibody typically will be formulated at a concentration of about0.1 mg/ml to 100 mg/ml, although wide variation outside of these rangesis permitted. For the treatment of disease, the appropriate dosage ofantibody or conjugate will depend on the type of disease to be treated,as defined above, the severity and course of the disease, whether theantibodies are administered for preventive or therapeutic purposes, thecourse of previous therapy, the patient's clinical history and responseto the antibody, and the discretion of the attending physician. Theantibody is suitably administered to the patient at one time or over aseries of treatments.

Depending on the type and severity of the disease, preferably from about1 mg/square meter to about 2000 mg/square meter of antibody is aninitial candidate dosage for administration to the patient, morepreferably from about 10 mg/square meter to about 1000 mg/square meterof antibody whether, for example, by one or more separateadministrations, or by continuous infusion. For repeated administrationsover several days or longer, depending on the condition, the treatmentis repeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful and are not excluded.

The present invention also includes kits comprising one or more of theelements described herein, and instructions for the use of thoseelements. In a preferred embodiment, a kit of the present inventionincludes antibody, antibody fragment or conjugate of the invention, anda therapeutic agent. The instructions for this preferred embodimentinclude instructions for inhibiting the growth of a cancer cell usingthe antibody, antibody fragment or conjugate of the invention, and thetherapeutic agent, and/or instructions for a method of treating apatient having a cancer using the antibody, antibody fragment orconjugate of the invention, and the therapeutic agent.

Preferably, the antibody used in the kit has the same amino acidsequence as the murine antibody EM164 produced by mouse hybridoma EM164(ATCC accession number PTA-4457), or the antibody is an epitope-bindingfragment thereof, wherein both the antibody and the fragmentspecifically bind to insulin-like growth factor-I receptor. The antibodyand antibody fragment used in the kit may also be a resurfaced versionof the EM164 antibody, a humanized version of the EM164 antibody, or analtered version of the EM164 antibody having least one nucleotidemutation, deletion or insertion. Antibodies and antibody fragments ofeach of these three versions retain the same binding specificity as theEM164 antibody.

Preferably, the therapeutic agent used in the kit is selected from thegroup consisting of docetaxel, paclitaxel, doxorubicin, epirubicin,cyclophosphamide, trastuzumab (Herceptin), capecitabine, tamoxifen,toremifene, letrozole, anastrozole, fulvestrant, exemestane, goserelin,oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab(Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan,etoposide, topotecan, gemcitabine, vinorelbine, estramustine,mitoxantrone, abarelix, zoledronate, streptozocin, rituximab (Rituxan),idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine,ibritumomab (Zevalin), tositumomab (Bexxar), interferon alpha-2b,melphalam, bortezomib (Velcade), altretamine, asparaginase, gefitinib(Iressa), erlonitib (Tarceva), anti-EGF receptor antibody (Cetuximab,Abx-EGF), and an epothilone. More preferably, the therapeutic agent is aplatinum agent (such as carboplatin, oxaliplatin, cisplatin), a taxane(such as paclitaxel, docetaxel), gemcitabine, or camptothecin.

The elements of the kits of the present invention are in a suitable formfor a kit, such as a solution or lyophilized powder. The concentrationor amount of the elements of the kits will be understood by the skilledartisan to varying depending on the identity and intended use of eachelement of the kit.

The cancers and cells therefrom referred to in the instructions of thekits include breast cancer, colon cancer, ovarian carcinoma,osteosarcoma, cervical cancer, prostate cancer, lung cancer, synovialcarcinoma, pancreatic cancer, melanoma, multiple myeloma, neuroblastoma,and rhabdomyosarcoma.

EXAMPLES

The invention is now described by reference to the following examples,which are illustrative only, and are not intended to limit the presentinvention.

Example 1 Murine EM164 Antibody

In this first example, the complete primary amino acid structure andcDNA sequence of a murine antibody of the present invention isdisclosed, together with its binding properties and means for itsexpression in recombinant form. Accordingly, there is provided a fulland complete disclosure of an antibody of the invention and itspreparation, such that one of ordinary skill in the immunological artswould be able to prepare said antibody without undue experimentation.

A. Generation of Anti-IGF-I Receptor Monoclonal Antibody Hybridoma

A cell line expressing human IGF-I receptor with a Y1251F mutation wasused for immunization as it expressed a high number of IGF-I receptors(˜10⁷ per cell). The Y1251F-mutation in the cytoplasmic domain of IGF-Ireceptor resulted in loss of transformation and anti-apoptoticsignaling, but did not affect IGF-I binding and IGF-I-stimulatedmitogenic signaling (O'Connor, R. et al., 1997, Mol. Cell. Biol., 17,427-435; Miura, M. et al., 1995, J. Biol. Chem., 270, 22639-22644). Themutation did not otherwise affect antibody generation because theantibody of this example bound to the extracellular domain of IGF-Ireceptor, which was identical for both the Y1251F mutant and the wildtype receptor.

A cell line expressing human IGF-I receptor with a Y1251F mutation wasgenerated from 3T3-like cells of a IGF-I-receptor-deficient mouse bytransfection with Y1251F-mutant human IGF-I-receptor gene together witha puromycin-resistance gene, and was selected using puromycin (2.5microgram/mL) and by FACS sorting for high IGF-I receptor expression(Miura, M. et al., 1995, J. Biol. Chem., 270, 22639-22644). A cell linehaving a high level of IGF-I receptor expression was further selectedusing a high concentration of puromycin such as 25 microgram/mL, whichwas toxic to most of the cells. Surviving colonies were picked and thosedisplaying a high level of IGF-I receptor expression were selected.

CAF1/J female mice, 6 months old, were immunized intraperitoneally onday 0 with Y125IF-mutant-human-IGF-I-receptor-overexpressing cells(5×10⁵ cells, suspended in 0.2 mL PBS). The animals were boosted with0.2 mL cell suspension as follows: day 2, 1×10⁶ cells; day 5, 2×10⁶cells; days 7, 9, 12, and 23, 1×10⁷ cells. On day 26, a mouse wassacrificed and its spleen removed.

The spleen was ground between two frosted glass slides to obtain asingle cell suspension, which was washed with serum-free RPMI mediumcontaining penicillin and streptomycin (SFM). The spleen cell pellet wasresuspended in 10 mL of 0.83% (w/v) ammonium chloride solution in waterfor 10 min on ice to lyse the red blood cells, and was then washed withserum-free medium (SFM). Spleen cells (1.2×10⁸) were pooled with myelomacells (4×10⁷) from the non-secreting mouse myeloma cell lineP3X63Ag8.653 (ATCC, Rockville, Md.; Cat. #CRL1580) in a tube, and washedwith the serum-free RPMI-1640 medium (SFM). The supernatant was removedand the cell pellet resuspended in the residual medium. The tube wasplaced in a beaker of water at 37° C. and 1.5 mL of polyethylene glycolsolution (50% PEG (w/v), average molecular weight 1500 in 75 mM HEPES,pH 8) was added slowly at a drop rate of 0.5 mL/minute while the tubewas gently shaken. After a wait of one minute, 10 mL of SFM was added asfollows: 1 mL over the first minute, 2 mL over the second minute, and 7mL over the third minute. Another 10 mL was then added slowly over oneminute. Cells were pelleted by centrifugation, washed in SFM andresuspended in RPMI-1640 growth medium supplemented with 5% fetal bovineserum (FBS), hypoxanthine/aminopterin/thymidine (HAT), penicillin,streptomycin, and 10% hybridoma cloning supplement (HCS). Cells wereseeded into 96-well flat-bottom tissue culture plates at 2×10⁵ spleencells in 200 μL per well. After 5-7 days, 100 μL per well were removedand replaced with growth medium supplemented with hypoxanthine/thymidine(HT) and 5% FBS. The general conditions used for immunization andhybridoma production were as described by J. Langone and H. Vunakis(Eds., Methods in Enzymology, Vol. 121, “Immunochemical Techniques, PartI”; 1986; Academic Press, Florida) and E. Harlow and D. Lane(“Antibodies: A Laboratory Manual”; 1988; Cold Spring Harbor LaboratoryPress, New York). Other techniques of immunization and hybridomaproduction can also be used, as are well known to those of skill in theart.

Culture supernatants from hybridoma clones were screened for binding topurified human IGF-I receptor by ELISA, for specific binding to cellsoverexpressing human IGF-I receptor, and for a lack of binding to cellsoverexpressing human insulin receptor by ELISA and FACS screening asdescribed below. Clones exhibiting higher binding affinity to cellsoverexpressing human IGF-I receptor than to cells overexpressing humaninsulin receptor were expanded and subcloned. The culture supernatantsof the subclones were further screened by the above binding assays. Bythis procedure, subclone 3F1-C8-D7 (EM164) was selected, and the heavyand light chain genes were cloned and sequenced as described below.

Human IGF-I receptor was isolated for use in the screening ofsupernatants from hybridoma clones for their binding to IGF-I receptorby the method below. Biotinylated IGF-I was prepared by modification ofrecombinant IGF-I using biotinylating reagents such assulfo-NHS-LC-biotin, sulfo-NHS-SS-biotin, or NHS-PEO₄-biotin.Biotinylated IGF-I was absorbed on streptavidin-agarose beads andincubated with lysate from cells that overexpressed human wild type orY1251F mutant IGFR. The beads were washed and eluted with a buffercontaining 2 to 4 M urea and detergent such as triton X-100 oroctyl-β-glucoside. Eluted IGF-I receptor was dialyzed against PBS andwas analyzed for purity by SDS-PAGE under reducing conditions, whichshowed alpha and beta chain bands of IGF-I receptor of molecular weightsabout 135 kDa and 95 kDa, respectively.

To check for the binding of hybridoma supernatants to purified IGF-Ireceptor, an Immulon-4HB ELISA plate (Dynatech) was coated with apurified human IGF-I receptor sample (prepared by dialysis fromurea/octyl-β-glucoside elution of affinity purified sample) diluted in50 mM CHES buffer at pH 9.5 (100 μL; 4° C., overnight). The wells wereblocked with 200 μL of blocking buffer (10 mg/mL BSA in TBS-T buffercontaining 50 mM Tris, 150 mM NaCl, pH 7.5, and 0.1% Tween-20) andincubated with supernatants from hybridoma clones (100 μL; diluted inblocking buffer) for about 1 h to 12 h, washed with TBS-T buffer, andincubated with goat-anti-mouse-IgG-Fc-antibody-horseradish peroxidase(HRP) conjugate (100 μL; 0.8 μg/mL in blocking buffer; JacksonImmunoResearch Laboratories), followed by washes and detection usingABTS/H₂O₂ substrate at 405 nm (0.5 mg/mL ABTS, 0.03% H₂O₂ in 0.1 Mcitrate buffer pH 4.2). Typically, a supernatant from a 3F1 hybridomasubclone yielded a signal of about 1.2 absorbance units within 3 min ofdevelopment, in contrast to values of 0.0 obtained for supernatants fromsome other hybridoma clones. General conditions for this ELISA weresimilar to the standard ELISA conditions for antibody binding anddetection as described by E. Harlow and D. Lane (“Using Antibodies: ALaboratory Manual”; 1999, Cold Spring Harbor Laboratory Press, NewYork), which conditions can also be used.

Screening of hybridoma supernatants for specific binding to human IGF-Ireceptor and not to human insulin receptor was performed using ELISA oncell lines that overexpressed human Y1251F-IGF-I receptor and on celllines that overexpressed human insulin receptor. Both cell lines weregenerated from 3T3-like cells of IGF-I receptor deficient mice. TheIGF-I receptor overexpressing cells and insulin receptor overexpressingcells were separately harvested from tissue culture flasks by quicktrypsin/EDTA treatment, suspended in growth medium containing 10% FBS,pelleted by centrifugation, and washed with PBS. The washed cells (100μL of about 1-3×10⁶ cells/mL) were added to wells of an Immulon-2HBplate coated with phytohemagglutinin (100 μL of 20 μg/mL PHA),centrifuged and allowed to adhere to PHA-coated wells for 10 min. Theplate with cells was flicked to remove PBS and was then dried overnightat 37° C. The wells were blocked with 5 mg/mL BSA solution in PBS for 1h at 37° C. and were then washed gently with PBS. Aliquots of thesupernatants from hybridoma clones (100 μL; diluted in blocking buffer)were then added to wells containing IGF-I-receptor-overexpressing cellsand to wells containing insulin receptor-overexpressing cells and wereincubated at ambient temperature for 1 h. The wells were washed withPBS, incubated with goat-anti-mouse-IgG-Fc-antibody-horseradishperoxidase conjugate (100 μL; 0.8 μg/mL in blocking buffer) for 1 h,followed by washes and then binding was detected using an ABTS/H₂O₂substrate. A typical supernatant from a 3F1 hybridoma subclone uponincubation with cells overexpressing IGF-I receptor yielded a signal of0.88 absorbance units within 12 min of development, in contrast to avalue of 0.22 absorbance units obtained upon incubation with cellsoverexpressing human insulin receptor.

The hybridoma was grown in Integra CL 350 flasks (Integra Biosciences,Maryland), according to manufacturer's specifications, to providepurified EM164 antibody. A yield of about 0.5-1 mg/mL antibody wasobtained in the harvested supernatants from the Integra flasks, based onquantitation by ELISA and by SDS-PAGE/Coomassie blue staining usingantibody standards. The antibody was purified by affinity chromatographyon Protein A-agarose bead column under standard purification conditionsof loading and washing in 100 mM Tris buffer, pH 8.9, containing 3 MNaCl, followed by elution in 100 mM acetic acid solution containing 150mM NaCl. The eluted fractions containing antibody were neutralized withcold 2 M K₂HPO₄ solution and dialyzed in PBS at 4° C. The concentrationof the antibody was determined by measuring absorbance at 280 nm(extinction coefficient=1.4 mg⁻¹ mL cm⁻¹). The purified antibody samplewas analyzed by SDS-PAGE under reducing conditions and Coomassie bluestaining, which indicated only heavy and light chain bands of antibodyat about 55 kDa and 25 kDa, respectively. The isotype of the purifiedantibody was IgG₁ with kappa light chain.

B. Binding Characterization of EM164 Antibody

The specific binding of the purified EM 164 antibody was demonstrated byfluorescence activated cell sorting (FACS) using cells overexpressinghuman IGF-I receptor and by using cells that overexpressed human insulinreceptor (FIG. 1). Incubation of EM 164 antibody (50-100 nM) in 100 μLcold FACS buffer (1 mg/mL BSA in Dulbecco's MEM medium) was performedusing cells overexpressing IGF-I receptor and using cells overexpressinginsulin receptor (2×10⁵ cells/mL) in a round-bottom 96-well plate for 1h. The cells were pelleted by centrifugation and washed with cold FACSbuffer by gentle flicking, followed by incubation withgoat-anti-mouse-IgG-antibody-FITC conjugate (100 μL; 10 μmg/mL in FACSbuffer) on ice for 1 h. The cells were pelleted, washed, and resuspendedin 120 μL of 1% formaldehyde solution in PBS. The plate was analyzedusing a FACSCalibur reader (BD Biosciences).

A strong fluorescence shift was obtained upon incubation of IGF-Ireceptor overexpressing cells with EM 164 antibody, in contrast to aninsignificant shift upon incubation of insulin receptor overexpressingcells with EM 164 antibody (FIG. 1), which demonstrated that the EM 164antibody was selective in its binding to IGF-I receptor and did not bindto insulin receptor. The control antibodies, anti-IGF-I receptorantibody 1H7 (Santa Cruz Biotechnology) and anti-insulin receptor alphaantibody (BD Pharmingen Laboratories), yielded fluorescence shifts uponincubations with cells that overexpressed IGF-I receptor and insulinreceptor, respectively (FIG. 1). A strong fluorescence shift was alsoobserved by FACS assay using EM 164 antibody and human breast cancerMCF-7 cells, which expressed IGF-I receptor (Dufourny, B. et al., 1997,J. Biol. Chem., 272, 31163-31171), which showed that EM164 antibodybound to human IGF-I receptor on the surface of human tumor cells.

The dissociation constant (K_(d)) for the binding of EM164 antibody withhuman IGF-I receptor was determined by ELISA titration of the binding ofantibody at several concentrations with either directly coated IGF-Ireceptor (affinity purified using biotinylated IGF-I, as above) orindirectly captured biotinylated IGF-I receptor. Biotinylated IGF-Ireceptor was prepared by biotinylation of detergent-solubilized lysatefrom IGF-I receptor overexpressing cells using PEO-maleimide-biotinreagent (Pierce, Molecular Biosciences), which was affinity purifiedusing an anti-IGF-I receptor beta chain antibody immobilized onNHS-agarose beads and was eluted with 2-4 M urea in buffer containingNP-40 detergent and dialyzed in PBS.

The K_(d) determination for the binding of EM164 antibody withbiotinylated IGF-I receptor was carried out by coating Immulon-2HBplates with 100 μL of 1 μg/mL streptavidin in carbonate buffer (150 mMsodium carbonate, 350 mM sodium bicarbonate) at 4° C. overnight. Thestreptavidin-coated wells were blocked with 200 μL of blocking buffer(10 mg/mL BSA in TBS-T buffer), washed with TBS-T buffer and incubatedwith biotinylated IGF-I receptor (10 to 100 ng) for 4 h at ambienttemperature. The wells containing indirectly captured biotinylated IGF-Ireceptor were then washed and incubated with EM164 antibody in blockingbuffer at several concentrations (5.1×10⁻¹³ M to 200 nM) for 2 h atambient temperature and were then incubated overnight at 4° C. The wellswere next washed with TBS-T buffer and incubated withgoat-anti-mouse-IgGH_(H+L)-antibody-horseradish peroxidase conjugate(100 μL; 0.5 μg/mL in blocking buffer), followed by washes and detectionusing ABTS/H₂O₂ substrate at 405 nm. The value of K_(d) was estimated bynon-linear regression for one-site binding.

A similar binding titration was carried out using the Fab fragment ofEM164 antibody, prepared by papain digestion of the antibody asdescribed by E. Harlow and D. Lane (“Using Antibodies: A LaboratoryManual”; 1999, Cold Spring Harbor Laboratory Press, New York).

The binding titration curve for the binding of EM164 antibody tobiotinylated human IGF-I receptor yielded a K_(d) value of 0.1 nM (FIG.2). The Fab fragment of EM164 antibody also bound the human IGF-Ireceptor very tightly with a K_(d) value of 0.3 nM, which indicated thatthe monomeric binding of the EM164 antibody to IGF-I receptor was alsovery strong.

This extremely low value of dissociation constant for the binding ofIGF-I receptor by EM164 antibody was in part due to a very slow off rateas verified by the strong binding signals observed after prolonged 1-2day washes of the antibody bound to immobilized IGF-I receptor.

EM164 antibody can be used for immunoprecipitation of IGF-I receptor, asdemonstrated by incubation of detergent-solubilized lysate of the humanbreast cancer MCF-7 cells with EM164 antibody immobilized on proteinG-agarose beads (Pierce Chemical Company). A Western blot of the EM164antibody immunoprecipitate was detected using a rabbit polyclonalanti-IGF-I receptor beta chain (C-terminus) antibody (Santa CruzBiotechnology) and a goat-anti-rabbit-IgG-antibody-horseradishperoxidase conjugate, followed by washes and enhanced chemiluminescence(ECL) detection. The Western blot of EM164 immunoprecipitate from MCF-7cells exhibited bands corresponding to the beta chain of IGF-I receptorat about 95 kDa and the pro-IGF-I receptor at about 220 kDa. Similarimmunoprecipitations were carried out for other cell types to checkspecies specificity of the binding of EM164 antibody, which also boundto IGF-I receptor from cos-7 cells (African green monkey), but did notbind to IGF-I receptor of 3T3 cells (mouse), CHO cells (Chinese hamster)or goat fibroblast cells (goat). The EM164 antibody did not detectSDS-denatured human IGF-I receptor in Western blots of lysates fromMCF-7 cells, which indicated that it bound to a conformational epitopeof native, non-denatured human IGF-I receptor.

The binding domain of EM164 antibody was further characterized using atruncated alpha chain construct, which comprised the cysteine richdomain flanked by L1 and L2 domains (residues 1-468) fused with the16-mer-C-terminus piece (residues 704-719) and which was terminated by aC-terminus epitope tag. This smaller IGF-I receptor, which lackedresidues 469-703, has been reported to bind IGF-I, although less tightlycompared to the native full-length IGF-I receptor (Molina, L. et al.,2000, FEBS Letters, 467, 226-230; Kristensen, C. et al., 1999, J. Biol.Chem., 274, 37251-37356). Thus, a truncated IGF-I receptor alpha chainconstruct was prepared comprising residues 1-468 fused to the C-terminuspiece that is residues 704-719 and flanked by a C-terminus myc epitopetag. A stable cell line which expressed this construct, and which alsoexpresses the construct transiently in human embryonic kidney 293Tcells, was constructed. A strong binding of EM164 antibody to thistruncated IGF-I receptor alpha chain construct was observed. Of the twoantibodies tested, IR3 (Calbiochem) also bound to this truncated alphachain, but 1H7 antibody (Santa Cruz Biotechnology) did not bind, whichindicated that the epitope of EM164 antibody was clearly distinct fromthat of 1H7 antibody.

C. Inhibition of Binding of IGF-I to MCF-7 Cells by EM164 Antibody

The binding of IGF-I to human breast cancer MCF-7 cells was inhibited byEM164 antibody (FIG. 3). MCF-7 cells were incubated with or without 5μg/mL EM164 antibody for 2 h in serum-free medium, followed byincubation with 50 ng/mL biotinylated IGF-I for 20 min at 37° C. Thecells were then washed twice with serum-free medium to remove unboundbiotin-IGF-I, and were then lysed in 50 mM HEPES, pH 7.4, containing 1%NP-40 and protease inhibitors. An Immulon-2HB ELISA plate was coatedwith a mouse monoclonal anti-IGF-I receptor beta chain antibody and wasused to capture the IGF-I receptor and bound biotin-IGF-I from thelysate. The binding of the coated antibody to the cytoplasmic C-terminaldomain of the beta chain of IGF-I receptor did not interfere with thebinding of biotin-IGF-I to the extracellular domain of IGF-I receptor.The wells were washed, incubated with streptavidin-horseradishperoxidase conjugate, washed again, and then detected using ABTS/H₂O₂substrate. The inhibition of IGF-I binding to MCF-7 cells by 5 μg/mLEM164 antibody was essentially quantitative, and was almost equivalentto that of the ELISA background obtained using a control lackingbiotin-IGF-I.

In addition to the assay described above for the inhibition of bindingof IGF-I to MCF-7 cells by EM164 antibody, the following assaydemonstrated that EM164 antibody was highly effective at displacingbound IGF-I from MCF-7 cells, as desired under physiological conditionsfor an antagonistic anti-IGF-I receptor antibody to displace the boundendogenous physiological ligand (such as IGF-I or IGF-II). In this IGF-Idisplacement assay, MCF-7 cells grown in a 12-well plate wereserum-starved and then incubated with biotinylated IGF-I (20-50 ng/mL)in serum-free medium at 37° C. (or at 4° C.) for 1 to 2 h. The cellswith bound biotinylated IGF-I were then treated with EM164 antibody or acontrol antibody (10-100 μg/mL) at 37° C. (or at 4° C.) for 30 min to 4h. Cells were then washed with PBS and lysed in lysis buffer containing1% NP-40 at 4° C. ELISA was carried out as described above to capturethe IGF-I receptor from the lysate and then detect the biotinylatedIGF-I bound to the receptor using streptavidin-horseradish peroxidaseconjugate. This ELISA demonstrated that EM164 antibody was able todisplace pre-bound biotinylated IGF-I from cells nearly completely (90%within 30 min and ˜100% within 4 h) at 37° C. and by about 50% in 2 h at4° C. In another experiment, NCI-H838 lung cancer cells were incubatedwith biotin-IGF-I, then washed and incubated with EM164 antibody at 4°C. for 2 h, which resulted in a 80% decrease in the bound biotin-IGF-I.Therefore, EM164 antibody was highly effective at displacing pre-boundIGF-I from cancer cells, which would be important therapeutically forthe antagonism of the IGF-I receptor by displacement of the boundendogenous physiological ligand.

The incubation of MCF-7 cells with EM164 antibody at 4° C. for 2 h (orat 37° C. for 30 min) did not result in a significant downregulation ofthe IGF-I receptor based on Western blot analysis using anti-IGF-Ireceptor beta chain antibody (Santa Cruz Biotechnology; sc-713),although a longer incubation with EM164 antibody at 37° C. for 2 hresulted in a 25% downregulation of the IGF-I receptor. Therefore, theinhibition of binding of IGF-I and the displacement of bound IGF-I byEM164 antibody at both 4° C. and 37° C. in these short-term experimentsmay not be explained by the down-regulation of the receptor due to thebinding of the EM164 antibody. The mechanism for the potent inhibitionof the binding of IGF-I to IGF-I receptor and for the displacement ofthe pre-bound IGF-I by EM164 antibody is likely to be competition forbinding, either through sharing of the binding site or through stericocclusion or through allosteric effects.

D. Inhibition of IGF-I Receptor Mediated Cell Signaling by EM164Antibody

Treatment of breast cancer MCF-7 cells and osteosarcoma SaOS-2 cellswith EM164 antibody almost completely inhibited intracellular IGF-Ireceptor signaling, as shown by the inhibition of IGF-I receptorautophosphorylation and by the inhibition of phosphorylation of itsdownstream effectors such as insulin receptor substrate-1 (IRS-1), Aktand Erk1/2 (FIGS. 4-6).

In FIG. 4, the MCF-7 cells were grown in a 12-well plate in regularmedium for 3 days, and were then treated with 20 μg/mL EM164 antibody(or anti-B4 control antibody) in serum-free medium for 3 h, followed bystimulation with 50 ng/mL IGF-I for 20 min at 37° C. The cells were thenlysed in ice-cold lysis buffer containing protease and phosphataseinhibitors (50 mM HEPES buffer, pH 7.4, 1% NP-40, 1 mM sodiumorthovanadate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 2.5mM EDTA, 10 μM leupeptin, 5 μM pepstatin, 1 mM PMSF, 5 mM benzamidine,and 5 μg/mL aprotinin). An ELISA plate was pre-coated with anti-IGF-Ireceptor beta chain C-terminus monoclonal antibody TC123 and wasincubated with the lysate samples for 5 h at ambient temperature tocapture IGF-I receptor. The wells containing the captured IGF-I receptorwere then washed and incubated with biotinylated anti-phosphotyrosineantibody (PY20; 0.25 μg/mL; BD Transduction Laboratories) for 30 min,followed by washes and incubation with streptavidin-horseradishperoxidase conjugate (0.8 μg/mL) for 30 min. The wells were washed anddetected with ABTS/H₂O₂ substrate. Use of a control anti-B4 antibodyshowed no inhibition of the IGF-I stimulated autophosphorylation ofIGF-I receptor. In contrast, a complete inhibition of the IGF-Istimulated autophosphorylation of IGF-I receptor was obtained upontreatment with EM164 antibody (FIG. 4).

To demonstrate inhibition of phosphorylation of insulin receptorsubstrate-1 (IRS-1), an ELISA using immobilized anti-IRS-1 antibody tocapture IRS-1 from lysates was used, followed by measurement of theassociated p85 subunit of phosphatidylinositol-3-kinase (PI-3-kinase)that binds to the phosphorylated IRS-1 (Jackson, J. G. et al., 1998, J.Biol. Chem., 273, 9994-10003). In FIG. 5, MCF-7 cells were treated with5 μg/mL antibody (EM164 or IR3) in serum-free medium for 2 h, followedby stimulation with 50 ng/mL IGF-I for 10 min at 37° C. Anti-IRS-1antibody (rabbit polyclonal; Upstate Biotechnology) was indirectlycaptured by incubation with coated goat-anti-rabbit-IgG antibody on anELISA plate, which was then used to capture IRS-1 from the cell lysatesamples by overnight incubation at 4° C. The wells were then incubatedwith mouse monoclonal anti-p85-PI-3-kinase antibody (UpstateBiotechnology) for 4 h, followed by treatment withgoat-anti-mouse-IgG-antibody-HRP conjugate for 30 min. The wells werethen washed and detected using ABTS/H₂O₂ substrate (FIG. 5). As shown inFIG. 5, EM164 antibody was more effective at inhibiting theIGF-I-stimulated IRS-1 phosphorylation than was IR3 antibody, and EM164antibody did not show any agonistic activity on IRS-1 phosphorylationwhen incubated with cells in the absence of IGF-I.

The activation of other downstream effectors, such as Akt and Erk1/2,was also inhibited in a dose-dependent manner by EM164 antibody inSaOS-2 cells (FIG. 6) and in MCF-7 cells, as was shown using Westernblots of lysates and phosphorylation-specific antibodies (rabbitpolyclonal anti-phospho-Ser⁴⁷³ Akt polyclonal and anti-phospho-ERK1/2antibodies; Cell Signaling Technology). A pan-ERK antibody demonstratedequal protein loads in all lanes (FIG. 6). Treatment of SaOS-2 cellswith EM164 antibody did not inhibit the EGF-stimulated phosphorylationof Erk1/2, thus demonstrating the specificity of inhibition of IGF-Ireceptor signaling pathway by EM164 antibody.

E. Inhibition of IGF-I-, IGF-II- and Serum-Stimulated Growth andSurvival of Human Tumor Cells by EM164 Antibody

Several human tumor cell lines were tested in serum-free conditions fortheir growth and survival response to IGF-I. These cell lines weretreated with EM164 antibody in the presence of IGF-I, IGF-II, or serum,and their growth and survival responses were measured using an Mu assayafter 2-4 days. Approximately 1500 cells were plated in a 96-well platein regular medium with serum, which was replaced with serum-free mediumthe following day (either serum-free RPMI medium supplemented withtransferrin and BSA, or phenol-red free medium as specified by Dufourny,B. et al., 1997, J. Biol. Chem., 272, 31163-31171). After one day ofgrowth in serum-free medium, the cells were incubated with about 75 μLof 10 μg/mL antibody for 30 min.-3 h, followed by the addition of 25 μLof IGF-I (or IGF-II or serum) solution to obtain a final concentrationof 10 ng/mL IGF-I, or 20 ng/mL IGF-II, or 0.04-10% serum. In someexperiments, the cells were stimulated first with IGF-I for 15 minbefore the addition of EM164 antibody, or both IGF-I and EM164 antibodywere added together. The cells were then allowed to grow for another 2-3days. A solution of MTT(3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; 25 μL ofa 5 mg/mL solution in PBS) was then added and the cells were returned tothe incubator for 2-3 h. The medium was then removed and replaced by 100μL DMSO, mixed, and the absorbance of the plate was measured at 545 nm.Several human tumor cell lines showed a growth and survival responseupon addition of IGF-I or IGF-II or serum that was significantlyinhibited by EM164 antibody, irrespective of whether the antibody wasadded before IGF-I, or if IGF-I was added before the antibody, or ifboth IGF-I and antibody were added together (Table 1). TABLE 1Inhibition of IGF-I-stimulated growth and survival of tumor cells byEM164 antibody % Inhibition by Fold growth response EM164 antibodyInhibition by to IGF-I (MTT assay: of IGF-I- EM164 antibody of ratio forIGF-I treated stimulated growth Growth/survival of vs untreated cells inin serum-free cells in 1.25-10% Tumor Cell Type serum-free medium)^(a)medium serum^(b) MCF-7 (breast) 1.7-2.8 100%  85% HT-3 (cervical) 2  70-90% ND Colo 205 (colon) 2.3 50% Yes HT-29 1.5 60% Yes NCI-H838 (lung)3   100%  85-90% Calu-6 1.6-1.8 85% Yes SK-LU-1 1.4 100%  No NCI-H5961.4 100%  Weakly A 549 1.2 80% ND A 375 (melanoma) 1.6 90% No SK-Mel-371.4 85% ND RD (rhabdomyocarcoma) 1.7  85-100% Yes SaOS-2 (osteosarcoma)2.5 100%  Yes A 431 (epidermoid) 2.2 85% Yes SK-N-SH (neuroblastoma) 2  85% 30-50%^(a)MTT assay of 3- to 4-day growth/survival of cells in response to 10ng/mL IGF-I in serum-free medium containing 5-10 μg/mL EM164 antibody.^(b)Inhibition of growth of cells in 1.25-10% serum in the presence of5-10 μg/mL EM164 antibody by MTT assay or colony formation assay basedon comparison with the control (with serum but without antibody); theextent of inhibition was quantitatively measured for MCF-7, NCI-H838 andSK-N-SH cells based on controls (without serum but# with antibody, and with serum but without antibody) to account forautocrine/paracrine IGF-stimulation by cells. ND indicates no data orpoor data due to staining difficulties.

The EM164 antibody strongly inhibited IGF-I-or serum-stimulated growthand survival of breast cancer MCF-7 cells (FIGS. 7 and 8). In a separateexperiment, the EM164 antibody strongly inhibited IGF-II-stimulatedgrowth and survival of MCF-7 cells. Previous reports using commerciallyavailable antibodies such as IR3 antibody showed only weak inhibition ofserum-stimulated growth and survival of MCF-7 cells, as confirmed inFIG. 7 for the IR3 and 1H7 antibodies (Cullen, K. J. et al., 1990,Cancer Res., 50, 48-53). In contrast, EM164 antibody was a potentinhibitor of the serum- or IGF-stimulated growth of MCF-7 cells. Asshown in FIG. 8, EM164 antibody was equally effective in inhibiting thegrowth and survival of MCF-7 cells over a wide range of serumconcentrations (0.04-10% serum).

The growth inhibition of MCF-7 cells by EM164 antibody was measured bycounting cells. Thus, in a 12-well plate, about 7500 cells were platedin RPMI medium with 10% FBS, in the presence or absence of 10 μg/mLEM164 antibody. After 5 days of growth, the cell count for the untreatedcontrol sample was 20.5×10⁴ cells, in contrast to a cell count of only1.7×10⁴ cells for the EM164 antibody-treated sample. Treatment with theEM164 antibody inhibited the growth of MCF-7 cells by about 12-fold in 5days. This inhibition by EM164 antibody was significantly greater thanwas a reported 2.5-fold inhibition using IR3 antibody in a 6-day assayfor MCF-7 cells (Rohlik, Q. T. et al., 1987, Biochem. Biophys. Res.Commun., 149, 276-281).

The IGF-I- and serum-stimulated growth and survival of a non-small celllung cancer line NCI-H838 were also strongly inhibited by EM164antibody, compared to a control anti-B4 antibody (FIG. 9). Treatmentwith EM164 antibody in serum-free medium produced a smaller signal thanthe untreated sample for both NCI-H838 and MCF-7 cells, presumablybecause EM 164 antibody also inhibited the autocrine and paracrine IGF-Iand IGF-II stimulation of these cells (FIGS. 7 and 9). The colony sizeof HT29 colon cancer cells was also greatly reduced upon treatment withEM164 antibody.

EM164 antibody is therefore unique among all known anti-IGF-I receptorantibodies in its effectiveness to inhibit the serum-stimulated growthof tumor cells such as MCF-7 cells and NCI-H838 cells by greater than80%.

The EM164 antibody caused growth arrest of cells in G0/G1 phase of cellcycle and abrogated the mitogenic effect of IGF-I. For cell cycleanalysis, MCF-7 cells were treated with IGF-I (20 ng/mL) in the presenceor absence of EM164 (20 μg/mL) for 1 day and then analyzed by propidiumiodide staining and flow cytometry. As shown in FIG. 25, the cycling ofcells in response to IGF-I stimulation in the absence of EM164 (with 41%of the cells in the S phase and 50% in the G0/G1 phase) was suppressedin EM164-treated cells (with only 9% in the S phase and 77% of the cellsin the G0/G1 phase).

In addition to its inhibition of cell proliferation, EM164 antibodytreatment resulted in apoptosis of cells. For measurement of apoptosis,cleavage of the cytokeratin CK18 protein by caspase was measured inNCI-H838 lung cancer cells incubated with IGF-I or serum in the presenceor absence of EM164 for 1 day (FIG. 26). In the absence of EM164, theaddition of IGF-I or serum resulted in a lower caspase-cleaved CK18signal compared to the no IGF-I control, indicating that IGF-I and serumprevent the activation of caspase. Treatment with EM164 suppressed theanti-apoptotic effects of IGF-I and serum, as indicated by the greatercleaved CK18 levels obtained in the presence of EM164 than in theabsence of EM164 (FIG. 26).

F. Synergistic Inhibition by EM164 Antibody of Growth and Survival ofHuman Tumor Cells in Combinations with Other Cytotoxic and CytostaticAgents

The combined administration of EM164 antibody with taxol wassignificantly more inhibitory to the growth and survival of non-smallcell lung cancer Calu6 cells than was taxol alone. Similarly, thecombination of EM164 antibody with camptothecin was significantly moreinhibitory than camptothecin alone toward the growth and survival ofcolon cancer HT29 cells. Because EM164 antibody alone was not expectedto be as toxic to cells as organic chemotoxic drugs, the synergismbetween the predominantly cytostatic effect of EM164 antibody and thecytotoxic effect of the chemotoxic drug may be highly efficacious incombination cancer therapies in clinical settings.

The combined effect of EM164 antibody with an anti-EGF receptor antibody(KS77) was significantly more inhibitory than either EM164 antibody orKS77 antibody alone on the growth and survival of several tumor celllines such as HT-3 cells, RD cells, MCF-7 cells, and A431 cells.Therefore, the synergistic effect of combining neutralizing antibodiesfor two growth factor receptors such as IGF-I receptor and EGF receptormay also be useful in clinical cancer treatment.

Based on the efficacy of EM164 antibody as a single agent in inhibitingthe proliferation and survival of diverse human cancer cell lines asshown in Table 1, additional efficacy studies were carried out usingcombinations of EM164 antibody with other anti-cancer therapeuticagents. In these studies on diverse cancer cell lines, the combinedtreatment of EM164 antibody and other anti-cancer therapeutic agentsresulted in an even greater anti-cancer efficacy than with either EM164or the other therapeutic agent alone. These combinations of EM164 withother therapeutic agents are therefore highly effective in inhibitingthe proliferation and survival of cancer cells. The therapeutic agentsthat can be combined with EM164 for improved anti-cancer efficacyinclude diverse agents used in oncology practice (Reference: Cancer,Principles & Practice of Oncology, DeVita, V. T., Hellman, S.,Rosenberg, S. A., 6th edition, Lippincott-Raven, Philadelphia, 2001),such as docetaxel, paclitaxel, doxorubicin, epirubicin,cyclophosphamide, trastuzumab (Herceptin), capecitabine, tamoxifen,toremifene, letrozole, anastrozole, fulvestrant, exemestane, goserelin,oxaliplatin, carboplatin, cisplatin, dexamethasone, antide, bevacizumab(Avastin), 5-fluorouracil, leucovorin, levamisole, irinotecan,etoposide, topotecan, gemcitabine, vinorelbine, estramustine,mitoxantrone, abarelix, zoledronate, streptozocin, rituximab (Rituxan),idarubicin, busulfan, chlorambucil, fludarabine, imatinib, cytarabine,ibritumomab (Zevalin), tositumomab (Bexxar), interferon alpha-2b,melphalam, bortezomib (Velcade), altretamine, asparaginase, gefitinib(Iressa), erlonitib (Tarceva), anti-EGF receptor antibody (Cetuximab,Abx-EGF), epothilones, and conjugates of cytotoxic drugs and antibodiesagainst cell-surface receptors.

For these combination therapies, EM164 is combined with one or-moreanti-cancer agents of diverse mechanisms of action such as alkylatingagents, platinum agents, hormonal therapies, antimetabolites,topoisomerase inhibitors, antimicrotubule agents, differentiationagents, antiangiogenic or antivascularization therapies, radiationtherapy, agonists and antagonists of leuteinizing hormone releasinghormone (LHRH) or gonadotropin-releasing hormone (GnRH), inhibitoryantibodies or small molecule inhibitors against cell-surface receptors,and other chemotherapeutic agents (Reference: Cancer, Principles &Practice of Oncology, DeVita, V. T., Hellman, S., Rosenberg, S. A., 6thedition, Lippincott-Raven, Philadelphia, 2001). In one example, thecombination of an LHRH antagonist antide (0.1 to 10 micromolar) andEM164 antibody (0.1 to 10 nanomolar) inhibited the proliferation ofMCF-7 breast cancer cells significantly more than that with either EM164or antide alone. In an example of a combination therapy with a platinumagent, the combined treatment with EM164 antibody (10 microgram/ml) andcisplatin (0.1-60 microgram/ml) resulted in a greater inhibition of theproliferation and survival of MCF-7 breast cancer cells in comparison tothe inhibition by either EM164 antibody or cisplatin alone.

These combinations of EM164 antibody with other therapeutic agents areeffective against several types of cancers including breast, lung,colon, prostate, pancreatic, cervical, ovarian, melanoma, multiplemyeloma, neuroblastoma, rhabdomyosarcoma and osteosarcoma. The EM164antibody and the therapeutic agent can be administered for cancertherapy either simultaneously or in sequence.

Conjugates of EM164 antibody with cytotoxic drugs are also valuable intargeted delivery of the cytotoxic drugs to the tumors overexpressingIGF-I receptor. Conjugates of EM164 antibody with radiolabels or otherlabels can be used in the treatment and imaging of tumors thatoverexpress IGF-I receptor.

G. Effect of EM164 Treatment, as a Single Agent or in Combination withAnti-Cancer Agents, in Human Cancer Xenografts in Immunodeficient Mice

Human non-small cell lung cancer Calu-6 xenografts were established inimmunodeficient mice by subcutaneous injections of 1×10⁷ Calu-6 cells.As shown in FIG. 10, these mice containing established 100 mm³ Calu-6xenografts were treated with EM 164 antibody alone (6 injections of 0.8mg/mouse, i. v., two per week) or with taxol alone (five injections oftaxol, i.p. every two days; 15 mg/kg), or with a combination of taxoland EM164 antibody treatments, or PBS alone (200 μL/mouse, 6 injections,two per week, i.v.) using five mice per treatment group. The growth oftumors was significantly slowed by EM164 antibody treatment compared toa PBS control. No toxicity of EM164 antibody was observed, based onmeasurements of the weights of the mice. Although taxol treatment alonewas effective until day 14, the tumor then started to grow back.However, the growth of the tumor was delayed significantly in the groupthat was treated by a combination of taxol and EM164 antibody, comparedto the group that was treated with taxol alone.

Human pancreatic cancer xenografts were established in 5 week-old,female SCID/ICR mice (Taconic) by subcutaneous injections of 10⁷ BxPC-3cells in PBS (day 0). The mice bearing established tumors of 80 mm³ werethen treated with EM164 alone (13 injections of 0.8 mg/mouse, i.v.,lateral tail vein, on days 12, 16, 19, 23, 26, 29, 36, 43, 50, 54, 58,61 and 64), with gemcitabine alone (two injections of 150 mg/kg/mouse,i.p., on days 12 and 19), with a combination of gemcitabine and EM164following the above schedules, PBS alone, and a control antibody alone(following the same schedule as EM164) using five mice in each of thefive treatment groups. As shown in FIG. 27, treatment with EM164 alone,or in combination with gemcitabine, resulted initially in totalregression of tumor xenografts in 4 of 5 animals in the EM164 treatmentgroup and in all 5 animals in the combination treatment group.Measurable tumor regrowth was only seen in more than one animal on day43 in the EM164 group and on day 68 in the combination treatment group,resulting in significantly smaller mean tumor volumes on day 74 incomparison with the control treatments (P=0.029 and 0.002, respectively;two-tailed T-test; FIG. 27). In another study, EM164 antibody treatment(alone or in combination with an anti-EGF receptor antibody;intraperitoneal injections) inhibited the growth of established BxPC-3xenografts in mice.

The murine EM164 and the humanized EM164 antibodies showed equivalentinhibition of the growth of established BxPC-3 xenografts in mice, thusdemonstrating that the potency of the humanized EM164 is equivalent tothat of the murine EM164 in vivo. In a comparison of different modes ofadministration of EM164 antibody, both intraperitoneal and intravenousadministrations of EM164 antibody showed equivalent inhibition of thegrowth of established BxPC-3 xenografts in mice. In another xenograftstudy, treatment with EM164 antibody showed significant growth delay ofestablished A-673 human rhabdomyosarcoma/Ewing's sarcoma xenografts inmice.

H. Cloning and Sequencing of the Light and Heavy Chains of EM164Antibody

Total RNA was purified from EM164 hybridoma cells. Reverse transcriptasereactions were performed using 4-5 μg total RNA and either oligo dT orrandom hexamer primers.

PCR reactions were performed using a RACE method described in Co et al.(J. Immunol., 148, 1149-1154 (1992)) and using degenerate primers asdescribed in Wang et al., (J. Immunol. Methods, 233, 167-177 (2000)).The RACE PCR method required an intermediate step to add a poly G tailon the 3′ ends of the first strand cDNAs. RT reactions were purifiedwith Qianeasy (Qiagen) columns and eluted in 50 μl 1×NEB buffer 4. A dGtailing reaction was performed on the eluate with 0.25 mM CoCl₂, 1 mMdGTP, and 5 units terminal transferase (NEB), in 1×NEB buffer 4. Themixture was incubated at 37° C. for 30 minutes and then ⅕ of thereaction (10 μl) was added directly to a PCR reaction to serve as thetemplate DNA.

The RACE and degenerate PCR reactions were identical except fordifferences in primers and template. The terminal transferase reactionwas used directly for the RACE PCR template, while the RT reaction mixwas used directly for degenerate PCR reactions.

In both RACE and degenerate PCR reactions the same 3′ light chainprimer: HindKL-tatagagctcaagcttggatggtgggaagatggatacagttggtgc (SEQ IDNO: 14) and 3′ heavy chain primer:

-   Bgl2IgGI-ggaagatctatagacagatgggggtgtcgttttggc (SEQ ID NO: 15) were    used.

In the RACE PCR, one poly C 5′ primer was used for both the heavy andlight chain:

-   EcoPolyC-TATATCTAGAATTCCCCCCCCCCCCCCCCC (SEQ ID NO: 16), while the    degenerate 5′ end PCR primers were:-   SacI MK-GGGAGCTCGAYATTGTGMTSACMCARWCTMCA (SEQ ID NO: 17) for the    light chain, and an equal mix of:-   EcoRIMH1-CTTCCGGAATTCSARGTNMAGCTGSAGSAGTC (SEQ ID NO: 18) and-   EcoRIMH2-CTTCCGGAATTCSARGTNMAGCTGSAGSAGTCWGG (SEQ ID NO: 19) for the    heavy chain.

In the above primer sequences, mixed bases are defined as follows:H=A+T+C, S=g+C, Y=C+T, K=G+T, M=A+C, R=A+g, W=A+T, V=A+C+G.

The PCR reactions were performed using the following program: 1) 94° C.3 min, 2) 94° C. 15 sec, 3) 45° C. 1 min, 4) 72° C. 2 min, 5) cycle backto step #2 29 times, 6) finish with a final extension step at 72° C. for10 min.

The PCR products were cloned into pBluescript II SK+ (Stratagene) usingrestriction enzymes created by the PCR primers.

Several individual light and heavy chain clones were sequenced byconventional means to identify and avoid possible polymerase generatedsequence errors (FIGS. 12 and 13). Using Chothia canonicalclassification definitions, the three light chain and heavy chain CDRswere identified (FIGS. 12-14).

A search of the NCBI IgBlast database indicated that the anti-IGF-Ireceptor antibody light chain variable region probably derived from themouse IgVk Cr1 germline gene while the heavy chain variable regionprobably derived from the IgVh J558.c germline gene (FIG. 15).

Protein sequencing of murine EM164 antibody was performed to confirm thesequences shown in FIGS. 12 and 13. The heavy and light chain proteinbands of purified EM164 antibody were transferred to a PVDF membranefrom a gel (SDS-PAGE, reducing conditions), excised from the PVDFmembrane and analyzed by protein sequencing. The N-terminal sequence ofthe light chain was determined by Edman sequencing to be: DVLMTQTPLS(SEQ ID NO:20), which matches the N-terminal sequence of the clonedlight chain gene obtained from the EM164 hybridoma.

The N-terminus of the heavy chain was found to be blocked for Edmanprotein sequencing. A tryptic digest peptide fragment of the heavy chainof mass 1129.5 (M+H+, monoisotopic) was fragmented via post-source decay(PSD) and its sequence was determined to be GRPDYYGSSK (SEQ ID NO:21).Another tryptic digest peptide fragment of the heavy chain of mass2664.2 (M+H+, monoisotopic) was also fragmented via post-source decay(PSD) and its sequence was identified as: SSSTAYMQLSSLTSEDSAVYYFAR (SEQID NO:22). Both of these sequences match perfectly those of CDR3 andframework 3 (FR3) of the cloned heavy chain gene obtained from the EM164hybridoma.

I. Recombinant Expression of EM164 Antibody

The light and heavy chain paired sequences were cloned into a singlemammalian expression vector (FIG. 16). The PCR primers for the humanvariable sequences created restriction sites that allowed the humansignal sequence to be attached while in the pBluescriptII cloningvector, and the variable sequences were cloned into the mammalianexpression plasmid using EcoRI and BsiWI or HindIII and ApaI sites forthe light chain or heavy chain, respectively (FIG. 16). The light chainvariable sequences were cloned in-frame onto the human IgK constantregion and the heavy chain variable sequences were cloned into the humanIggamma1 constant region sequence. In the final expression plasmids,human CMV promoters drove the expression of both the light and heavychain cDNA sequences. Expression and purification of the recombinantmouse EM164 antibody proceeded according to methods that are well-knownin the art.

Example 2 Humanized Versions of EM164 Antibody

Resurfacing of the EM164 antibody to provide humanized versions suitableas therapeutic or diagnostic agents generally proceeds according to theprinciples and methods disclosed in U.S. Pat. No. 5,639,641, and asfollows.

A. Surface Prediction

The solvent accessibility of the variable region residues for a set ofantibodies with solved structures was used to predict the surfaceresidues for the murine anti-IGF-I receptor antibody (EM164) variableregion. The amino acid solvent accessibility for a set of 127 uniqueantibody structure files (Table 2) were calculated with the MC softwarepackage (Pedersen et al., 1994, J. Mol. Biol., 235, 959-973). The tenmost similar light chain and heavy chain amino acid sequences from thisset of 127 structures were determined by sequence alignment. The averagesolvent accessibility for each variable region residue was calculated,and positions with greater than a 30% average accessibility wereconsidered to be surface residues. Positions with averageaccessibilities of between 25% and 35% were further examined bycalculating the individual residue accessibility for only thosestructures with two identical flanking residues. TABLE 2 127 antibodystructures from the Brookhaven database used to predict the surface ofanti-IGF-I-receptor antibody (EM164) 127 Brookhaven structure files usedfor surface predictions 2rcs 3hfl 3hfm 1aif 1a3r 1bbj 43c9 4fab 6fab7fab 2gfb 2h1p 2hfl 1a6t 1axt 1bog 2hrp 2jel 2mcp 2pcp 1yuh 2bfv 2cgr8fab 1ae6 1bvl 2dbl 2f19 2fb4 2fbj 1sm3 1tet 1vfa glb2 1a4j 1cly 1vge1yec 1yed 1yee 1nsn 1opg 1osp 1aj7 1ay1 1clz 1plg 1psk 1rmf 1sbs 1ncd1nfd 1ngp 1acy 1afv 1cbv 1nld 1nma 1nmb 1nqb 1mcp 1mfb 1mim 15c8 1a5f1axs 1mlb 1mpa 1nbv 1ncb 1jrh 1kb5 1kel 1ap2 1b2w 1adq 1kip 1kir 1lve1mam 1igi 1igm 1igt 1ad0 1baf 1cfv 1igy 1ikf 1jel 1jhl 1gpo 1hil 1hyx1a0q 1bjm 1clo 1iai 1ibg 1igc 1igf 1fpt 1frg 1fvc 1aqk 1bln 1d5b 1gaf1ggi 1ghf 1gig 1fai 1fbi 1fdl 1ad9 1bbd 1f58 1fgv 1fig 1flr 1for 1dbl1dfb 1a3l 1bfo 1eap 1dsf 1dvfB. Molecular Modeling:

A molecular model of murine EM164 was generated using the OxfordMolecular software package AbM. The antibody framework was built fromstructure files for the antibodies with the most similar amino acidsequences, which were 2jel for the light chain and lnqb for the heavychain. The non-canonical CDRs were built by searching a C-α structuredatabase containing non-redundant solved structures. Residues that liewithin 5 Å of a CDR were determined.

C. Human Ab Selection

The surface positions of murine EM164 were compared to the correspondingpositions in human antibody sequences in the Kabat database (Johnson, G.and Wu, T. T. (2001) Nucleic Acids Research, 29: 205-206). The antibodydatabase management software SR (Searle 1998) was used to extract andalign the antibody surface residues from natural heavy and light chainhuman antibody pairs. The human antibody surface with the most identicalsurface residues, with special consideration given to positions thatcome within 5 Å of a CDR, was chosen to replace the murine anti-IGF-Ireceptor antibody surface residues.

D. PCR Mutagenesis

PCR mutagenesis was performed on the murine EM164 cDNA clone (above) tobuild the resurfaced, human EM164 (herein huEM164). Primer sets weredesigned to make the 8 amino acid changes required for all testedversions of huEM164, and additional primers were designed toalternatively make the two 5 Å residue changes (Table 3). PCR reactionswere performed with the following program: 1) 94° C. 1 min, 2) 94° C. 15sec, 3) 55° C. 1 min, 4) 72° C. 1 min, 5) cycle back to step #2 29times, 6) finish with a final extension step at 72° C. for 4 min. ThePCR products were digested with their corresponding restriction enzymesand were cloned into the pBluescript cloning vectors as described above.Clones were sequenced to confirm the desired amino acid changes. TABLE 3PCR primers used to build 4 humanized EM164 antibodies Primer SequenceSEQ ID NO: Em164hcvv CAGGTGTACACTCCCAGGTCCAACTG 23GTGCAGTCTGGGGCTGAAGTGGTGAA GCCTG Em164hcqqgo11CAATCAGAAGTTCCAGGGGAAGGCCA 24 CAC Em164hcqqgo12CCTTCCCCTGGAACTTTCTGATTGTAG 25 TTAGTACG Em164lcv3CAGGTGTACACTCCGATGTTGTGATG 26 ACCCAAACTCC Em164lc13CAGGTGTACACTCCGATGTTTTGATG 27 ACCCAAACTCC Em164lcp18GACTAGATCTGCAAGAGATGGAGGCT 28 GGATCTCCAAGAC Em164lcbg12TTGCAGATCTAGTCAGAGCATAGTAC 29 ATAGTAATG Em164r45GAATGGTACCTGCAGAAACCAGGCCA 30 GTCTCCAAGGCTCCTGATCTAC Em164a67o11GTGGCAGTGGAGCAGGGACAGATTTC 31 AC Em164a67o12 GAAATCTGTCCCTGCTCCACTGCCAC32 TGE. Variable Region Surface Residues

The antibody resurfacing techniques described by Pedersen et al. (J.Mol. Biol., 235, 959-973, 1994) and Roguska et al. (Protein Eng., 9,895-904, 1996) begin by predicting the surface residues of the murineantibody variable sequences. A surface residue is defined as an aminoacid that has at least 30% of its total surface area accessible to awater molecule.

The 10 most homologous antibodies in the set of 127 antibody structurefiles were identified (FIGS. 17 and 18). The solvent accessibility foreach Kabat position was averaged for these aligned sequences and thedistribution of the relative accessibilities for each residue were asshown in FIG. 19. Both the light and heavy chain have 26 residues withaverage relative accessibilities of at least 30% (FIG. 19): theseresidues were therefore the predicted surface residues for EM164.Several residues had average accessibilities of between 25% and 35%, andthese were further examined by averaging only the antibodies with twoidentical residues flanking either side of the residue (Tables 4 and 5).After this additional analysis, the original set of surface residuesthat was identified above remained unchanged. TABLE 4 Surface residuesand average accessibility (ave. acc.) for the light and heavy chainvariable sequences of EM164 antibody EM164 Surface Residues Light ChainHeavy Chain EM164 Kabat # Ave. Acc. EM164 Kabat # Ave. Acc. D 1 45.89 Q1 58.19 L 3 41.53 Q 3 34.08 T 7 31.40 Q 5 34.36 L 9 50.08 A 9 38.01 L 1535.45 L 11 47.72 Q 18 39.79 K 13 46.51 R 24 34.36 P 14 31.49 S 26 32.63G 15 31.42 Q 27 34.35 K 19 34.41 N 28 36.38 K 23 31.23 P 40 43.05 T 2836.24 G 41 46.56 P 41 44.01 Q 42 34.92 G 42 42.62 K 45 30.58 Q 43 46.85S 52 30.40 E 61 46.68 S 56 41.46 K 62 44.87 G 57 42.41 K 64 38.92 D 6045.96 R 65 40.06 S 67 38.20 K 73 35.92 R 77 42.61 S 74 48.91 E 81 38.46S 82B 32.72 V 95E 34.83 S 84 35.21 K 103 31.10 E 85 39.62 K 107 36.94 D98 36.00 R 108 60.13 A 106 37.65 A 109 53.65 S 113 43.42

TABLE 5 Borderline Surface Residues Light Chain Heavy Chain EM164 Kabat# Ave. Acc. EM164 Kabat # Ave. Acc. T 5 28.68 Q 3 31.62 T 7 30.24 Q 536.07 P 12 26.59 P 14 29.88 G 16 25.20 G 15 30.87 D 17 25.73 S 17 25.64S 20 25.37 K 19 35.06 R 24 36.73 K 23 31.48 S 26 31.00 G 26 30.53 Q 2732.29 S 31 27.12 S 27A 29.78 R 56 NA V 27C 29.05 T 68 27.71 V 29 NA T 7024.65 Q 42 34.92 S 75 18.80 K 45 32.24 S 82B 32.87 S 52 30.02 P 97 NA R54 29.50 Y 99 NA D 70 26.03 V 103 NA R 74 NA T 111 25.95 E 79 26.64 A 8029.61 V 95E 42.12 G 100 29.82 K 103 31.10 E 105 25.78Residues which had average accessibilities between 25% and 35% werefurther analyzed by averaging a subset of antibodies that had twoidentical residues flanking either side of the residue in question.These borderline surface positions and their new average accessibilitiesare given. The NA's refer to residues with no identical flankingresidues in the 10 most similar antibodies.F. Molecular Modeling to Determine which Residues Fall within 5 Å of aCDR

The molecular model above, generated with the AbM software package, wasanalyzed to determine which EM164 surface residues were within 5 Å of aCDR. In order to resurface the murine EM164 antibody, all surfaceresidues outside of a CDR should be changed to the human counterpart,but residues within 5 Å of a CDR are treated with special care becausethey may also contribute to antigen specificity. Therefore, these latterresidues must be identified and carefully considered throughout thehumanization process. The CDR definitions used for resurfacing combinethe AbM definition for heavy chain CDR2 and Kabat definitions for theremaining 5 CDRs (FIG. 14). Table 6 shows the residues that were within5 Å of any CDR residue in either the light or heavy chain sequence ofthe EM164 model. TABLE 6 EM164 antibody framework surface residueswithin 5 Å of a CDR EM164 Surface Residues within 5 Å of a CDR Lightchain Heavy chain D1 T28 L3 K73 T7 S74 P40 Q42 K45 G57 D60 E81G. Identification of the Most Homologous Human Surfaces

Candidate human antibody surfaces for resurfacing EM164 were identifiedwithin the Kabat antibody sequence database using SR software, whichprovided for the searching of only specified residue positions againstthe antibody database. To preserve the natural pairings, surfaceresidues of both the light and heavy chains were compared together. Themost homologous human surfaces from the Kabat database were aligned inrank order of sequence identity. The top 5 surfaces are given in Table7. These surfaces were then compared to identify which of them wouldrequire the least changes within 5Å of a CDR. The Leukemic B-cellantibody, CLL 1.69, required the least number of surface residue changes(10 in total) and only two of these residues were within 5Å of a CDR.

The full length variable region sequence for EM164 was also alignedagainst the Kabat human antibody database and CLL 1.69 was againidentified as the most similar human variable region sequence. Together,these sequence comparisons identified the human Leukemic B-cell antibodyCLL 1.69 as the preferred choice as a human surface for EM164. TABLE 7The top 5 human sequences extracted from the Kabat database 5 MostHomologous Human Antibody Surfaces Antibody Light Chain SEQ ID NO:MuEM164 D L TL L Q PG Q K G DS R EK K R A 33 CLL1.69 D V T L L P P G Q RG D A R E K K R — 34 MSL5 D Q S L I P P G Q K G D S R D K K R A 35CDP571 D M S S V R P G Q K G S S S D K K R — 36 LC3aPB E V S G P R P G QR G D S R E K K R — 37 SSbPB E V S G P R P G Q R G D S R E K K R — 38Antibody Heavy Chain SEQ ID NO: MuEM164 Q Q Q A L K P G K K TP G Q E K KR K SS S E A S 39 CLL1.69 Q Q V A V K P G K K T P G Q Q K Q G K S S S EQ S 40 MSL5 Q Q Q P L K P G K K T P G K D D K G T S N N E Q S 41 CDP571Q Q V A V K P G K K T P G Q Q K K G K S S S E Q S 42 LC3aPB — Q V A V KP G K K T P G Q Q K Q G K S S S E Q S 43 SSbPB — Q V A V K P G K K T P GQ Q K Q G E S S S E Q S 44Alignments were generated by SR (Pedersen 1993). The EM164 surfaceresidues that come within 5Å of a CDR are underlined.H. Construction of Humanized EM164 Genes

The ten surface residue changes for EM164 (Table 7) were made using PCRmutagenesis techniques as described above. Because eight of the surfaceresidues for CLL 1.69 were not within 5Å of a CDR, these residues werechanged from murine to human in all versions of humanized EM164 (Tables8 and 9). The two light chain surface residues that were within 5Å of aCDR (Kabat positions 3 and 45) were either changed to human or wereretained as murine. Together, these options generate the four humanizedversions of EM164 that were constructed (FIGS. 22 and 23).

Of the four humanized versions, version 1.0 has all 10 human surfaceresidues. The most conservative version with respect to changes in thevicinity of the CDR is version 1.1, which retained both of the murinesurface residues that were within 5Å of a CDR. All four humanized EM164antibody genes were cloned into an antibody expression plasmid (FIG. 16)for use in transient and stable transfections. TABLE 8 Residue changesfor versions 1.0-1.3 of humanized EM164 antibody Changes in all versionsLight muQ18 to huP18; muS67 to huA67 Chain: Heavy muQ5 to huV5; muL11 tohuV11; muE61 to muK64 to Chain: huQ61; huQ64; muR65 to huG65; muA106 tohuQ106 huEM164 changes Light Chain aa3 Light Chain aa45 +TC,25/32 Total5A Mu hu mu hu Mouse Res v1.0 V R 0 v1.1 L K 2 v1.2 L R 1 v1.3 V K 1I. Comparison of the Affinities of Humanized EM164 Antibody Versionswith Murine EM164 Antibody for Binding to Full-Length IGF-I Receptor andto Truncated IGF-I Receptor Alpha Chain

The affinities of the humanized EM164 antibody versions 1.0-1.3 werecompared to those of murine EM164 antibody through binding competitionassays using biotinylated full-length human IGF-I receptor ormyc-epitope tagged truncated IGF-I receptor alpha chain, as describedabove. Humanized EM164 antibody samples were obtained by transienttransfection of the appropriate expression vectors in human embryonickidney 293T cells, and antibody concentrations were determined by ELISAusing purified humanized antibody standards. For ELISA bindingcompetition measurements, mixtures of humanized antibody samples andvarious concentrations of murine EM164 antibody were incubated withindirectly captured biotinylated full-length IGF-I receptor ormyc-epitope tagged truncated IGF-I receptor alpha chain. Afterequilibration, the bound humanized antibody was detected using agoat-anti-human-Fab′₂-antibody-horseradish peroxidase conjugate. Plotsof ([bound murine Ab]/[bound humanized Ab]) vs ([murine Ab]/[humanizedAb]), which theoretically yield a straight line withslope=(K_(d humanized Ab)/K_(d murine Ab)), were used to determine therelative affinities of the humanized and murine antibodies.

An exemplary competition assay is shown in FIG. 11. An Immulon-2HB ELISAplate was coated with 100 μL of 5 μg/mnL streptavidin per well incarbonate buffer at ambient temperature for 7 h. The streptavidin-coatedwells were blocked with 200 μL of blocking buffer (10 mg/mL BSA in TBS-Tbuffer) for 1 h, washed with TBS-T buffer and incubated withbiotinylated IGF-I receptor (5 ng per well) overnight at 4° C. The wellscontaining indirectly captured biotinylated IGF-I receptor were thenwashed and incubated with mixtures of humanized EM164 antibody (15.5 ng)and murine antibody (0 ng, or 16.35 ng, or 32.7 ng, or 65.4 ng, or 163.5ng) in 100 μL blocking buffer for 2 h at ambient temperature and werethen incubated overnight at 4° C. The wells were then washed with TBS-Tbuffer and incubated with goat-anti-human-Fab′₂-antibody-horseradishperoxidase conjugate for 1 h (100 μL; 1 μg/mL in blocking buffer),followed by washes and detection using ABTS/H₂O₂ substrate at 405 nm.

The plot of ([bound murine Ab]/[bound humanized Ab]) vs ([murineAb]/[humanized Ab]) yielded a straight line (r²=0.996) with slope(=K_(d humanized Ab)/K_(d murine Ab)) of 0.52. The humanized antibodyversion 1.0 therefore bound to IGF-I receptor more tightly than didmurine EM164 antibody. Similar values for the gradient, ranging fromabout 0.5 to 0.8, were obtained for competitions of versions 1.0, 1.1,1.2 and 1.3 of humanized EM164 antibodies with murine EM164 antibody forbinding to full-length IGF-I receptor or to truncated IGF-I receptoralpha chain, which indicated that all of the humanized versions of EM164antibody had similar affinities, which were all better than that of theparent murine EM164 antibody. A chimeric version of EM164 antibody with92F→C mutation in heavy chain showed a slope of about 3 in a similarbinding competition with murine EM164 antibody, which indicated that the92F→C mutant of EM 164 had a 3-fold lower affinity than did murine EM164antibody for binding to IGF-I receptor. The humanized EM164 v1.0antibody showed a similar inhibition of IGF-I-stimulated growth andsurvival of MCF-7 cells as did the murine EM164 antibody (FIG. 24). Theinhibition of serum-stimulated growth and survival of MCF-7 cells byhumanized EM164 v1.0 antibody was similar to the inhibition by murineEM164 antibody. TABLE 9 Segment Light Chain Heavy Chain FR1 1-23 (withan occasional residue 1-30 (with an occasional at 0, and a deletion at10 in residue at 0) V_(λ) chains) CDR1 24-34 (with possible insertions31-35 (with possible numbered as 27A, B, C, D, E, F) insertions numberedas 35A, B) FR2 35-49 36-49 CDR2 50-56 50-65 (with possible insertionsnumbered as 52A, B, C) FR3 57-88 66-94 (with possible insertionsnumbered as 82A, B, C) CDR3 89-97 (with possible insertions 95-102 (withpossible numbered as 95A, B, C, D, E, F) insertions numbered as 100A, B,C, D, E, F, G, H, I, J, K) FR4 98-107 (with a possible insertion 103-113numbered as 106A)The Kabat numbering system is used for the light chain and heavy chainvariable region polypeptides of the different versions of the EM164 Ab.The amino acid residues are grouped into Framework (FR) andComplementarity Determining Regions (CDR) according to position in thepolypeptide chain.Taken from Kabat et al. Sequences of Proteins of Immunological Interest,Fifth Edition, 1991, NIH Publication No. 91-3242J. Process of Providing Improved Anti-IGF-I-Receptor Antibodies Startingfrom the Murine and Humanized Antibody Sequences Described Herein:

The amino acid and nucleic acid sequences of the anti-IGF-I receptorantibody EM164 and its humanized variants were used to develop otherantibodies that have improved properties and that are also within thescope of the present invention. Such improved properties includeincreased affinity for the IGF-I receptor. Several studies have surveyedthe effects of introducing one or more amino acid changes at variouspositions in the sequence of an antibody, based on the knowledge of theprimary antibody sequence, on its properties such as binding and levelof expression (Yang, W. P. et al., 1995, J. Mol. Biol., 254, 392-403;Rader, C. et al., 1998, Proc. Natl. Acad. Sci. USA, 95, 8910-8915;Vaughan, T. J. et al., 1998, Nature Biotechnology, 16, 535-539).

In these studies, variants of the primary antibody have been generatedby changing the sequences of the heavy and light chain genes in theCDR1, CDR2, CDR3, or framework regions, using methods such asoligonucleotide-mediated site-directed mutagenesis, cassettemutagenesis, error-prone PCR, DNA shuffling, or mutator-strains of E.coli (Vaughan, T. J. et al., 1998, Nature Biotechnology, 16, 535-539;Adey, N. B. et al., 1996, Chapter 16, pp. 277-291, in “Phage Display ofPeptides and Proteins”, Eds. Kay, B. K. et al., Academic Press). Thesemethods of changing the sequence of the primary antibody have resulted,through the use of standard screening techniques, in improved affinitiesof such secondary antibodies (Gram, H. et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 3576-3580; Boder, E. T. et al., 2000, Proc. Natl. Acad.Sci. USA, 97, 10701-10705; Davies, J. and Riechmann, L., 1996,Immunotechnolgy, 2, 169-179; Thompson, J. et al., 1996, J. Mol. Biol.,256, 77-88; Short, M. K. et al., 2002, J. Biol. Chem., 277, 16365-16370;Furukawa, K. et al., 2001, J. Biol. Chem., 276, 27622-27628).

By a similar directed strategy of changing one or more amino acidresidues of the antibody, the antibody sequences described in thisinvention can be used to develop anti-IGF-I receptor antibodies withimproved functions, such as antibodies having suitable groups such asfree amino groups or thiols at convenient attachment points for covalentmodification for use, for example, in the attachment of therapeuticagents.

K. Alternative Expression System for Murine, Chimeric and OtherAnti-IGF-I Receptor Antibodies

The murine anti IGF-I receptor antibody was also expressed frommammalian expression plasmids similar to those used to express thehumanized antibody (above). Expression plasmids are known that havemurine constant regions including the light chain kappa and heavy chaingamma-1 sequences (McLean et al., 2000, Mol Immunol., 37, 837-845).These plasmids were designed to accept any antibody variable region,such as for example the murine anti-IGF-I receptor antibody, by a simplerestriction digest and cloning. Additional PCR of the anti-IGF-Ireceptor antibody was usually required to create the restrictioncompatible with those in the expression plasmid.

An alternative approach for expressing the fully murine anti-IGF-Ireceptor antibody was to replace the human constant regions in thechimeric anti-IGF-I receptor antibody expression plasmid. The chimericexpression plasmid (FIG. 16) was constructed using cassettes for thevariable regions and for both the light and heavy chain constantregions. Just as the antibody variable sequences were cloned into thisexpression plasmid by restriction digests, separate restriction digestswere used to clone in any constant region sequences. The kappa lightchain and gamma-I heavy chain cDNAs were cloned, for example, frommurine hybridoma RNA, such as the RNA described herein for cloning ofthe anti-IGF-I antibody variable regions. Similarly, suitable primerswere designed from sequences available in the Kabat database (see Table10). For example, RT-PCR was used to clone the constant region sequencesand to create the restriction sites needed to clone these fragments intothe chimeric anti-IGF-I receptor antibody expression plasmid. Thisplasmid was then used to express the fully murine anti-IGF-I receptorantibody in standard mammalian expression systems such as the CHO cellline. TABLE 10 Primers designed to clone the murine gamma-1 constantregion and murine kappa constant region respectively. Murine ConstantRegion Primers Primer name Primer Sequence SEQ ID NO: MuIgG1 C3endXTTTTGAGCTCTTATTTACCAGGA 45 GAGTGGGAGAGGCTCTT MuIgG1 C5endHTTTTAAGCTTGCCAAAACGACAC 46 CCCCATCTGTCTAT MuIgKap C3endBTTTTGGATCCTAACACTCATTCC 47 TGTTGAAGC MuIgKap C5endETTTTGAATTCGGGCTGATGCTGC 48 ACCAACTGThe primers were designed from sequences available in the Kabat database(Johnson, G and Wu, T.T. (2001) Nucleic Acids Research, 29: 205-206).

Statement of Deposit

The hybridoma that makes murine EM164 antibody was deposited with theAmerican Type Culture Collection, PO Box 1549, Manassas, Va. 20108, onJun. 14, 2002, under the Terms of the Budapest Treaty and assigned ATCCaccession number PTA-4457.

Certain patents and printed publications have been referred to in thepresent disclosure, the teachings of which are hereby each incorporatedin their respective entireties by reference.

While the invention has been described in detail and with reference tospecific embodiments thereof, it will be apparent to one of skill in theart that various changes and modifications can be made thereto withoutdeparting from the spirit and scope thereof.

1.-31. (canceled)
 32. An IGF-IR antagonist comprising a recombinantsingle-chain antibody capable of effectively inhibiting IGF-IR-agonistinteraction.
 33. An IGF-IR antagonist according to claim 32, wherein,the antibody is a chimeric single-chain recombinant antibody.
 34. AnIGF-IR antagonist according to claim 33, wherein the antibody is ahumanized single-chain recombinant antibody.
 35. An IGF-IR single-chainrecombinant antibody capable of effectively inhibiting IGF-IR-agonistinteraction wherein the recombinant antibody consists essentially of aV_(L) domain antigen binding portion linked to a V_(H) domain antigenbinding portion through a peptide linker.
 36. A recombinant antibodyaccording to claim 35, wherein said antigen binding portions of saidV_(L) and V_(H) domains are murine antibody domains.
 37. A recombinantantibody according to claim 35, wherein the antibody consistsessentially of a V_(L) domain antigen binding portion linked through apeptide linker to a V_(H) domain antigen binding portion linked to animmunoglobulin constant domain.
 38. A recombinant antibody according toclaim 37, wherein said V_(L) and V_(H) domains are murine antibodydomains.
 39. A recombinant antibody according to claim 37, wherein saidimmunoglobulin constant domain comprises C_(H2) and C_(H3) regions of aconstant domain.
 40. A recombinant antibody according to claim 39,wherein said C_(H2) and C_(H3) regions are from a non-murine mammalianimmunoglobulin domain.
 41. A recombinant antibody according to claim 40,wherein said C_(H2) and C_(H3) regions are human immunoglobulin regions.